In this randomized trial, Saul Rajak et al. compare silk sutures (removed at 7–10 days) or absorbable sutures (left in place) during surgery for the management of trachomatous trichiasis. Trachoma causes blindness through an anatomical abnormality called trichiasis (lashes touching the eye). Trichiasis can recur after corrective surgery. We tested the hypothesis that using absorbable sutures instead of silk sutures might reduce the risk of recurrent disease among patients with major trichiasis in a randomised trial.1,300 individuals with major trichiasis from rural villages in the Amhara Region of Ethiopia were recruited and assigned (1∶1) by computer-generated randomisation sequence to receive trichiasis surgery using either an absorbable suture (polyglactin-910) or silk sutures (removed at 7–10 days) in an otherwise identical surgical technique. Participants were examined every 6 months for 2 years by clinicians masked to allocation. The primary outcome measure was recurrent trichiasis (≥one lash touching the eye) at 1 year. There was no difference in prevalence of recurrent trichiasis at 1 year . The two groups also did not differ in terms of corneal opacification, visual acuity, conjunctival inflammation, and surgical complications.There was no evidence that use of absorbable polyglactin-910 sutures was associated with a lower prevalence of trichiasis recurrence at 1 year postsurgery than silk sutures. However, from a programmatic perspective, polyglactin-910 offers the major advantage that patients do not have to be seen soon after surgery for suture removal. The postoperative review after surgery using absorbable polyglactin-910 sutures can be delayed for 3–6 months, which might allow us to better determine whether a patient needs additional surgery.ClinicalTrials.govNCT00522860Please see later in the article for the Editors' Summary Chlamydia trachomatis. This bacterium is spread through contact with infected eye secretions or with contaminated towels or clothes, and by flies. Recurrent infections with C. trachomatis during early childhood cause inflammation of the tissue lining the eye lid , which can lead to conjunctival scarring. If this scarring is severe, the eyelids turn inwards (entropion) and the lashes rub across the eye's surface (the cornea). This condition—trachomatous trichiasis—is extremely painful and, if not treated with surgery, can lead to irreversible corneal opacities and visual impairment by middle age. It is estimated that 8 million people have trichiasis and that an additional 8 million people are blind or visually impaired as a result of the condition.Globally, around 40 million people—mostly people living in rural areas in developing countries where there are water shortages, poor personal hygiene, and crowded living conditions—have active trachoma, an infectious eye disease that is caused by Surgery for trichiasis, antibiotics for infection, and facial cleanliness and environmental improvements to reduce transmission together constitute the SAFE strategy for the control of blinding trachoma. Unfortunately, trichiasis recurs in nearly two-thirds of patients within 3 years of surgery, often within the first year. How the surgery is performed, its quality, and the severity of entropion and conjunctival scarring at the time of surgery all contribute to trichiasis recurrence. In this randomized trial (a study in which randomly chosen groups of patients receive different treatments for a disease and are followed to compare the outcomes of these interventions), the researchers investigate whether using absorbable sutures instead of silk sutures reduces the risk of recurrent disease among patients with major trichiasis (more than five lashes touching the cornea). Sutures are used to sew up surgical incisions. Silk sutures, which are used routinely during trichiasis surgery, have to be removed 7–10 days after surgery when the incision may not have stably healed. Absorbable sutures might provide more stable fixation of the eye tissue while healing is taking place and might, therefore, reduce the recurrence of trichiasis after surgery.The researchers randomly assigned 1,300 people living in Ethiopia (the country with the highest rates of trachoma and trichiasis) to receive trichiasis surgery using silk sutures (removed at 7–10 days) or absorbable sutures (left in place); the other details of the surgery were identical for all the patients. The trial's primary outcome was recurrent trichiasis (one or more lash touching the eye) at 1 year. Secondary outcomes included the rate of recurrence, visual acuity, corneal opacity, and conjunctival inflammation at 2 years, and surgical complications. At 1 year, 18.2% of the patients in the absorbable suture group and 19.7% in the silk suture group had developed recurrent trichiasis. That is, the prevalence of recurrence in the two groups was similar. There was also no difference in the rate of trichiasis recurrence between the groups 2 years after surgery. Moreover, the two groups did not differ in terms of corneal opacity and visual acuity, conjunctival inflammation, or surgical complications.These findings provide no evidence to suggest that the use of absorbable sutures during trichiasis surgery is clinically better than the use of silk sutures. However, the researchers note that the use of absorbable sutures would eliminate the need for patients to return to the clinic soon after their operation to have their sutures removed. In remote rural settings, it can be difficult for patients, who are often elderly and poor, to attend clinics. Thus, it might be better for trachoma services to concentrate on encouraging patients to return 3–6 months after surgery when the need for additional surgery can be determined rather than trying to encourage them to come back after 7–10 days for suture removal. The use of absorbable sutures would also avoid any complications arising from patients failing to come back to have their stitches removed. The researchers suggest, therefore, that trachoma control programs should now consider the potential logistical advantages of using absorbable sutures rather than silk sutures during trichiasis surgery despite the lack of any apparent clinical difference between the two suture types.http://dx.doi.org/10.1371/journal.pmed.1001137.Please access these Web sites via the online version of this summary at PLoS Medicine Research Article by Saul Rajak et al. describes another randomized trial undertaken by these researchers that compares surgery and epilation  for the treatment of trichiasis in EthiopiaAn accompanying trachoma , including details of the Alliance for Global Elimination of Trachoma by the year 2020 (GET 2020) and a personal story about blinding trachomaThe World Health Organization has information on trachomaThe UK National Health Service Choices website also provide information on Orbis, an international nonprofit organization devoted to blindness prevention and treatment in developing countries, provides information about trachomaInternational Trachoma Initiative provides detailed information about trachoma and a personal story about trichiasis surgery in EthiopiaThe Global Atlas of Trachoma is an open-access resource on the geographical distribution of trachomaThe Light for the World is a nonprofit organization dedicated to ensuring the rights of persons with disabilities in developing countries, including people in Ethiopia with trachoma Chlamydia trachomatis infection in early childhood provoke chronic conjunctival inflammation (active trachoma). This inflammation leads to scarring of conjunctival tissue, which causes the eyelids to roll in (entropion) and the lashes to scratch the surface of the eye (trachomatous trichiasis [TT]). Ultimately, blinding corneal opacification (CO) develops from the persistent abrasion of lashes and secondary bacterial infection.Trachoma is the leading infectious cause of blindness worldwide Blinding trachoma is reported to be prevalent in more than 50 countries To prevent blindness from trichiasis, surgery is performed to correct the entropion, lifting the eyelashes off the cornea. Ophthalmic services are generally limited in trachoma endemic settings; therefore, specially trained nurses usually perform the surgery in the community. Several different procedures have been tried over the last century Several factors contribute to recurrent trichiasis, which can be divided into early surgery-related and later disease-related factors. Data from prospective studies suggest that the majority of recurrent trichiasis develops within 6 mo of surgery, indicating the importance of how the surgery is performed Suture type, positioning, and tension are likely to be important aspects of surgical technique that contribute to TT recurrence. Silk sutures are used as standard in trichiasis surgery and need to be removed 7–10 d postoperatively A retrospective review of surgical outcomes in Egypt found that recurrence rates were significantly lower in individuals who had received long-lasting absorbable sutures (0.8%) compared to those with silk sutures (43.5%) The National Health Research Ethics Review Committee of the Ethiopian Ministry of Science and Technology, the London School of Hygiene and Tropical Medicine Ethics Committee, and Emory University Institutional Review Board approved the trial. Potential participants were provided with both written and oral information in Amharic about the trial. For those agreeing to participate, written informed consent in Amharic was required prior to enrolment. If the participant was unable to read and write, the information sheet and consent form were read to them and their consent recoded by witnessed thumbprint. An independent Data Safety Monitoring Committee reviewed the trial for patient safety and there were no deviations from the original protocol. No interim analyses for efficacy or futility were planned or conducted. The Trial Protocol is described in kebele) in the study area were trained to recognize trichiasis and visited each village in their kebele to identify patients.Eligible participants were individuals aged 18 y or over with previously unoperated major trichiasis  who presented during a TT surgical treatment campaign in rural villages in the West Gojjam zone of the Amhara Region of Ethiopia from March to June 2008. Exclusion criteria were previous eyelid surgery, medically unfit, or pregnancy (self-reported or clearly evident). The surgical campaigns were advertised in local markets, churches, and schools. Additionally, health extension workers from the subdistricts . The testing distance was reduced to 2 or 1 m if necessary. For those unable to read the LogMAR chart at 1 m their visual acuity was tested by counting fingers at 1 m or hand movements at 0.5 m. For visual acuities of counting fingers or less, LogMAR values were attributed: counting fingers, 2.0; hand movements, 2.5; perception of light, 3.0; no perception of light, 3.5. No patients had spectacles for distance correction. Ophthalmic examinations were conducted in a darkened room using 2.5× magnification loupes and a bright torch. A single ophthalmologist (SNR) performed all baseline examinations. The number of lashes touching the eye was counted (“lash burden”) and also subdivided by the part of the eye contacted when looking straight ahead: cornea, lateral conjunctiva, or medial conjunctiva. Clinical evidence of epilation was identified by the presence of broken or newly growing lashes, or areas of absent lashes. Upper lid entropion was graded by assessing the degree of inward rotation of the eyelid margin . The degFollowing the preoperative clinical assessment, participants were randomised to one of two surgical intervention groups, which differed only in the type of sutures that were used. The posterior lamella tarsal rotation procedure was used in all cases Five nurses, who had previously been trained in and were regularly performing TT surgery, performed the surgery in this trial. They were selected as the best surgeons from a larger group of nine during a 2-d standardisation workshop. The training was conducted by an experienced Ethiopian ophthalmologist (ABK), who had contributed to the development of the WHO TT surgeon certification manual All participants were seen 7–10 d postoperatively, when silk sutures were removed and any complications noted and treated as needed. Follow-up assessments were conducted at 3, 6, 12, 18, and 24 mo postoperatively. On each occasion an ophthalmic examination was performed and digital photographs taken. Photographs were not taken at 3 mo. Visual acuity was measured at 12 and 24 mo. The 12-mo and 24-mo follow-up examinations were conducted by the same single ophthalmologist (SNR) who performed the preoperative examinations; the 3-, 6-, and 18-mo examinations were conducted by a single ophthalmic nurse (EH). Neither of these examiners was involved in performing surgery in this trial. The examiners were standardised to each other and showed strong agreement between these two observers for the presence of trichiasis in a preliminary assessment of 200 eyes (kappa = 0.86) Several different ophthalmic nurses who did not take part in subsequent follow-ups conducted the 7-d follow-up. Any individual who developed significant recurrent trichiasis during the follow-up period was offered repeat surgery performed by a senior surgeon. These reoperated individuals continued to be followed up according to the trial protocol. Individuals in whom other ophthalmic pathology  was detected were referred to the regional ophthalmic services.The primary outcome measure was the proportion of those individuals seen at the 12-mo follow-up who were found to have either (1) recurrent trichiasis, defined as one or more lashes touching the eye or clinical evidence of epilation, or (2) a history of repeat TT surgery during the first year. Individuals not seen at 12 mo were excluded from this primary analysis. A priori defined secondary outcome measures at 12 and 24 mo were: CO, visual acuity, entropion, and conjunctival inflammation. Change in CO was determined both from direct comparison of the 1- and 2-y photographs with the baseline photographs and from the field grading scores. This enabled us to detect progression or regression using an alternative method of assessment of corneal opacities.Participants were randomly allocated to the silk or polyglactin-910 suture arms using a 1∶1 allocation ratio for each surgeon, using a computer-generated randomisation sequence with random block sizes. Randomisation was stratified by surgeon because of possible intersurgeon variability (each surgeon had a separate sequence). The statistician held the master randomisation lists. The random allocation sequences for each surgeon were concealed in sequentially numbered, sealed, opaque envelops, which were placed in separate containers for each surgeon; a person independent of all other aspects of the trial prepared these envelopes. On most days during the surgery campaign two or three surgeons operated simultaneously. Following the baseline examination, participants were allocated to the next available surgeon. A trained field worker was responsible for implementing the intervention assignment in a dedicated area, separated from those performing the preoperative examinations. The field worker and surgeon jointly confirmed the suture allocation and recorded this in the surgical logbook. The same field worker dispensed the appropriate sutures to the surgeon. Participants and surgeons were aware of the suture allocation. The two individuals  who were responsible for all the clinical observations in this trial were masked to the allocation. For the purposes of the trial analysis only one eye per person was considered. If the participant had bilateral trichiasis operated on, the study eye  was chosen at random using a computer generated randomisation list.Using a conservative 1-y risk of TT recurrence of 10% in the silk arm we considered a 50% reduction in recurrence to 5% in the polyglactin-910 arm to be programmatically significant Data were double entered into an Access (Microsoft) database and transferred to Stata 11 (StataCorp) for analysis. The analysis was on an intention-to-treat basis. For participants who had bilateral surgery only the randomly designated study eye was included in the analysis. We made every effort to obtain all outcome data for every participant. We excluded patients from analyses if they had missing data relevant to a particular analysis, as indicated in the results. Missing data were not imputed.p<0.2) were retained in the multivariable model. The surgeon with the lowest recurrence rate at 2 y was used as the reference for the others in the models.The primary outcome (recurrent trichiasis at 12 mo) and secondary outcomes were compared between the two groups using logistic regression analyses to estimate the odds ratio (OR) and 95% CI. Time to recurrence was analysed with Kaplan-Meier survival curves, and Cox regression models were used to estimate hazard ratios (HRs) and associated 95% CI. Individuals who had reached endpoint at a previous time point or were permanently lost to follow-up from that time onwards were censored. A multivariable logistic regression model was developed to estimate OR and 95% CI for associations of any recurrent trichiasis during the 2 y of follow-up with potential explanatory factors. Variables that were associated with recurrent trichiasis on univariate analysis  .p = 0.60) or the number of participants seen at each follow-up (Primary outcome data are available for 608/650 (93.5%) participants in the silk group and 628/650 (96.7%) of participants in the polyglactin-910 group . Overallollow-up . All 1,3Baseline sociodemographic characteristics were balanced by randomisation group . All parTrichiatic lashes touched the cornea in 1,049 (80.7%) eyes . CoexistAt 12-mo follow-up, recurrent trichiasis was present in 221 study eyes and a further 13 participants had undergone repeat surgery since baseline. Prevalence of recurrence was similar in the two arms of the trial . The risk difference for any recurrent trichiasis between the silk and polyglactin-910 arms was −0.016 (95% CI −0.059 to 0.023). The possibility of effect modification between arm and other factors on the primary outcome was investigated; none were significant, including surgeon.There was no evidence of a difference in the rate of recurrence between the two arms of the trial during the 2-y follow-up period  . Of the p = 0.21; p = 0.045) and suture fragments in situ .Complications were observed in 219 participants (16.9%). The overall complication risk was similar in the two arms of the trial  reduce the recurrence of trichiasis a year after surgery, compared with standard silk sutures removed at 7 to 10 d. We found no evidence of a difference in recurrence risk at either the primary endpoint (1 y) or over the 2-y follow-up period. The risk difference between arms in the primary end point was very small (1.1%) with narrow confidence intervals (−3.9% to 6.0%). This finding is consistent with the two sutures being equivalent from a clinical perspective, although the trial was not designed to test this hypothesis.The relationship between suture type and trichiasis recurrence was examined in a nonrandomised study from Egypt, which found a significantly lower recurrence rate in cases where absorbable sutures were used Our results suggest that, despite the lack of difference in recurrence risk, there are operational reasons why a trachoma control program might consider using absorbable sutures such as polyglactin-910. Firstly, most individuals requiring TT surgery live in relatively remote rural settings, and many are relatively old and with limited financial resources. Under such conditions transport may be limited or a significant financial burden, presenting a major challenge for the patient to reattend a distant health facility for suture removal. The use of absorbable sutures would eliminate the need for an early postoperative review at 7 to 10 d, allowing the service to focus on a more important 3- to 6-mo review to check for recurrent trichiasis and provide repeat surgery. Secondly, because not all patients will attend for routine follow-up after surgery, retained sutures are not uncommon and can cause significant trauma to the cornea. Even with the considerable logistical resources (vehicles and personnel) available in this trial and the high level of engagement by the participants in the follow-up, we still found that 2.6% of people in the silk arm had unremoved sutures at 3 mo. It is likely that this problem is significantly more frequent under operational conditions. The use of absorbable sutures generally avoids this problem. Whether a trachoma control program adopts the use of absorbable sutures for trichiasis surgery may be influenced by several locally specific considerations, including the relative local costs of different sutures types and the potential cost and time savings of not having to arrange for early review and suture removal.The trichiasis recurrence risk in our trial is at the lower end of the range typically reported; 19%  of the trial eyes reexamined at 1 y had recurrent TT. Most trials or consecutive series have reported similar 1-y TT recurrence risks in the range of about 20% to 40% at 1 y The reasons for recurrence are multiple. The time profile of recurrence suggests that there are two distinct phases: (1) early recurrence, occurring during the first 6 mo; (2) late recurrence, which occurs from 6 mo onwards and develops at a reduced rate. In the few prospective trials or case series with a year or more follow-up, early recurrence has tended to account for the majority of cases We found that recurrent trichiasis at both 1 and 2 y was significantly associated with both more severe entropion at baseline and baseline conjunctival inflammation. Several studies have found a consistent relationship between the severity of preoperative disease  and the risk of recurrence The type of operation performed also has a major effect on the outcome. A systematic review of the alternative procedures that have been compared in clinical trials found evidence to support the use of procedures that involve a full thickness incision through the tarsal plate, rotation of the distal fragment, and fixation with everting sutures The quality of the surgery is a major determinant of outcome. We found significant variation in the recurrence risk between surgeons. Surgeons were assigned to operate on patients on a “first-come-first-served” basis; therefore, it is unlikely that there was selection bias in the severity of cases operated on by specific surgeons in this trial. Moreover, intersurgeon variation persisted after adjusting for baseline disease severity in the model. Several other studies have found significant variation between surgeons, suggesting that there might be subtle variations in technique that influence the risk of recurrence Chlamydia trachomatis infection between participants in the different trials Late recurrent TT might occur owing to the immuno-fibrogenic factors that caused conjunctival scarring and TT in the first place, rather than problems directly related to the surgical procedure Complications of trichiasis surgery are uncommon. There were a few cases with moderate bleeding during surgery, all of whom were managed successfully with firm pressure on the eyelid for a few minutes. Similarly, postoperative wound infections were uncommon and resolved rapidly with antibiotics. The only notable differences between the two trial arms occurred later: retained suture and conjunctival granulomas (large enough to obscure vision) were more common in lids where silk sutures had been used. Silk is recognised to be quite proinflammatory in operative wounds, which might explain the higher granulomas rate.This study has a number of strengths including the large size, high follow-up rates, good balance of clinical and demographic characteristics between the randomisation groups, the fact that observations at each time point were made by a single observer, and that outcome measures were determined by masked individuals. There were slightly more individuals in the polyglactin-910 group who were lost to follow-up at 12 mo; however, most of these participants had moved out of the study area and so this small difference is not attributable to a treatment effect. Despite careful standardisation procedures, there was still some variation between the results of the different surgeons, indicating the importance of how the surgery is performed. These may include subtle variation in the length of the incision or the tension on the everting sutures. Individuals with minor trichiasis were excluded from this trial and enrolled into a separate trial evaluating the efficacy of surgery versus epilation. However, we are unaware of any reason to think that these findings could not be generalised to the full range of trichiasis severity. It would be interesting to know if there were significant differences in the participants' experience of the different sutures used in this trial and their perspectives on the potential benefit of not needing to have the absorbable sutures removed; however, we did not formally investigate this. Detecting differences in changes in both corneal disease and visual acuity would probably require a longer follow-up period.Polyglactin-910 absorbable sutures had a similar risk of trichiasis recurrence to silk sutures for PLTR TT surgery and comparable secondary outcomes. However, from a programmatic perspective, polyglactin-910 offers the major advantage that patients do not have to be seen soon after surgery for suture removal. The postoperative review can be delayed for 3–6 mo, which might allow us to better determine who needs additional surgery and offset the slightly higher cost of absorbable sutures. The logistical advantages of using absorbable sutures should be taken into consideration when considering the choice of suture material.Table S1(a) Expanded grading system for entropion, conjunctivilisation and CO. (b) Conjunctivilisation: anterioplacement of the muco-cutaneous junction of the upper eyelid. (c) Corneal scarring (assessed with eye in primary position).(DOCX)Click here for additional data file.Text S1Trial protocol.(DOC)Click here for additional data file.Text S2CONSORT checklist.(DOC)Click here for additional data file.


Objective. To compare two internal fixation devices clinically in stabilisation of intertrochanteric femur fractures. Methods. Eighty-seven patients were randomised upon their admission to the hospital using a sealed envelope method. Forty-five were treated with proximal femur nail antirotation (PFNA) and 42 with reverse less invasive stabilisation system (LISS). The perioperative data were recorded and compared in relation to fracture type. Results. In each type of fractures, no significant differences were found with respect to the blood loss, the quality of reduction, the time to bony healing, and the Harris hip score between the 2 groups. The mean duration of surgery was significantly longer in reverse LISS group than in PFNA group. Conclusion. Both the PFNA and the reversed LISS are effective in the treatment of different types of intertrochanteric femur fractures. PFNA is superior to reverse LISS in terms of surgical time, weight-bearing, and perhaps fluoroscopy time. Numerous internal fixation devices have been used to stabilise intertrochanteric femur fractures, and they can be divided into 2 categories: extramedullary fixation devices and intramedullary fixation devices. It is generally accepted that dynamic hip screw (DHS) is the implant of choice in the treatment of stable intertrochanteric femur fractures AO/OTA 31-A1) [-A1 [1]. Between September 2010 and August 2011, 100 patients with 100 intertrochanteric femoral fractures were randomised upon their admission to the hospital using a sealed envelope method. The inclusion criteria were ages above 65. Those with pathological fractures, osteoarthritis of the hips, and ASA [All patients were evaluated preoperatively with use of two standard plain radiographs, an anterior-posterior radiograph, and a medial-lateral radiograph. Surgical treatment was performed as soon as the patient's general medical condition allowed. Prophylactic intravenous first generation cephalosporin was administered half an hour before operation and discontinued 48–72 hours postoperatively. Internal fixation was performed by 3 orthopaedic consultants  and full weight-bearing (P < 0.001) were significant earlier in group I than in group II. The fluoroscopy time was significantly longer in group II than in group I. No significant difference was found with respect to the time of hospital stay.In type 31 A1 fractures, both the time to begin with partial weight-bearing . PFNA also shortened fluoroscopy time, but not statistically significant in unstable fractures (31A2 and 31A3). This can be explained that we are very familiar with PFNA [The study population and the baseline data  were similar in each fracture type between the 2 groups. The most important finding of this study was that PFNA could significantly shorten surgical time compared with reverse LISS (31A1, ith PFNA  and lackGood results were achieved with both the reverse LISS and PFNA in each fracture type, which was in accordance with the findings by Zhou et al.  and Han A weakness of this study is that we are familiar with PFNA but not with reverse LISS, for it is originally designed for distal femur. Another weakness is the relatively small patient group. Further studies are required concerning LISS application to proximal femur. In conclusion, the results of the present study show that both the PFNA and the reverse LISS provide effective methods of treatment for intertrochanteric hip fractures. PFNA is superior to reverse LISS in terms of surgical time, weight-bearing, and perhaps fluoroscopy time. Mechanical failure can be minimized when the rehabilitation program is made based on individual characteristics.


RNA polymerase II (PolII) transcribes RNA within a chromatin context, with nucleosomes acting as barriers to transcription. Despite these barriers, transcription through chromatin in vivo is highly efficient, suggesting the existence of factors that overcome this obstacle. To increase the resolution obtained by standard chromatin immunoprecipitation, we developed a novel strategy using micrococcal nuclease digestion of cross-linked chromatin. We find that the chromatin remodeler Chd1 is recruited to promoter proximal nucleosomes of genes undergoing active transcription, where Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover. The expression of a dominant negative form of Chd1 results in increased stalling of PolII past the entry site of the promoter proximal nucleosomes. We find that Chd1 evicts nucleosomes downstream of the promoter in order to overcome the nucleosomal barrier and enable PolII promoter escape, thus providing mechanistic insight into the role of Chd1 in transcription and pluripotency.DOI:http://dx.doi.org/10.7554/eLife.02042.001 DNA is tightly packaged in a material called chromatin inside the cell nucleus. To produce proteins this DNA must first be transcribed to produce a molecule of messenger RNA, which is then translated to make a protein. To assist with this process cells ‘unpack’ certain regions of the DNA so that enzymes that catalyze the different steps in this process can have access to the DNA.A protein called Chd1 is involved in the unpacking process in yeast, but its role in more complex animals is not clear. Now, Skene et al. have shown that this protein is needed to allow the enzyme that catalyzes the transcription of DNA—an enzyme called RNA polymerase II—to do its job. Chd1 acts to unpack the tightly packaged DNA from chromatin, thus allowing the transcription of the DNA to proceed. In the absence of Chd1 activity, RNA polymerase II stalls at the gene promoter—the region of DNA that starts the transcription of a particular gene. This work highlights how the packaging of DNA in the cell is highly dynamic and controls fundamental biological processes.Skene et al. modified a well-known genetic technique called ChIP-seq. Previous ChIP-seq protocols typically provided a blurry, low-resolution map of where proteins bound to chromatin. Skene et al. used an enzyme to ‘chew back’ the DNA to reveal the exact ‘footprints’ of the Chd1 protein and the RNA polymerase II enzyme on the chromatin in mice. It will be possible to adapt this new protocol to map the positions of other proteins, which will help to improve our understanding of the ways in which chromatin regulates access to DNA.DOI:http://dx.doi.org/10.7554/eLife.02042.002 The eukaryotic genome is packaged into chromatin, with the basic unit being a nucleosome, consisting of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Chromatin limits the accessibility of DNA-binding factors to the underlying DNA sequence and presents a strong physical barrier that must be overcome by RNA polymerase II (PolII) during transcription. In vitro experiments have shown that transcription initiation is the most affected and that elongation is also highly inefficient unless nucleosomes are destabilized . DespiteThrough the combined action of PolII, elongation factors, nucleosome modifications and chromatin remodelers, the chromatin barrier to transcription is highly dynamic, with nucleosomal turnover correlating with gene expression . NucleosAlthough the intrinsic structural preferences of DNA contribute substantially to nucleosome occupancy and positioning in vivo, this chromatin landscape is further manipulated by chromatin remodelers . Chromat. In contrast, in the gene body, Chd1 reassembles nucleosomes and suppresses histone turnover. Interestingly, mammalian Chd1 is required for pluripotency of embryonic stem (ES) cells by maintaining euchromatin and is also required for efficient reprogramming, suggesting that Chd1 plays a key role in mammalian cellular identity  were transfected with a transgene expressing FLAG-tagged wild-type mouse Chd1 or a mutant variant harboring the replacement of a conserved lysine to an arginine residue (K510R) in the ATP-binding site of the remodeler . The K51osophila . In composophila .10.7554/Micrococcal nuclease (MNase) digestion of cross-linked chromatin to increase the resolution of ChIP-seq. MNase digests unprotected DNA, leaving DNA fragments corresponding to mono-nucleosomes and sub-nucleosomal fragments, presumably protected by proteins cross-linked to the extra-nucleosomal DNA . We found that under native conditions only 14% of Chd1 could be extracted . This leomal DNA . By usinomal DNA . By usinomal DNA . This teInitially analysis of all the recovered fragments was performed. As expected, wild-type Chd1 was recruited to the 5′ end of genes, similar to what has been shown for endogenous Chd1 in mouse ES cells . FurtherSaccharomyces cerevisiae, where deletion of Chd1 had no effect on the +1 nucleosome occupancy but only the surrounding nucleosomes, blocking Chd1 activity in MEFs affected promoter proximal nucleosomes including the +1 nucleosome .Chromatin remodelers are known to have a major role in the correct positioning and occupancy of nucleosomes . Therefocleosome . In S. ccleosome . Howevercleosome . TherefoThe decrease in nucleosome occupancy was most apparent at genes with the highest PolII levels at the promoter, as expected given that Chd1 occupancy correlates with PolII promoter occupancy, with no statistically significant change at genes with low PolII levels at the promoter . Genes wTo establish whether the effects of the K510R mutant can be mimicked by reduced levels of Chd1, endogenous levels of Chd1 were reduced using a short-hairpin RNA . NucleosThe mapping of MNase-digested mono-nucleosomes provides information regarding steady-state nucleosome occupancy and positioning, but does not yield insight regarding nucleosome dynamics. We used CATCH-IT  to measure nucleosome turnover . CATCH-IWe found that nucleosome turnover in the gene body correlated with increasing levels of elongating PolII . This isIn contrast to nucleosome turnover at the gene body, nucleosome turnover at the promoter was decreased upon the expression of K510R-Chd1. We sought to determine if this reduction correlated with PolII promoter density given that both Chd1 occupancy and loss in nucleosome occupancy was most pronounced at genes with high PolII promoter density. Genes with higher levels of PolII displayed increased nucleosome turnover surrounding the TSS , as mighS. cerevisiae PHO5 gene promoter indicated that Chd1 was required to remove the +1 nucleosome, which occludes the TSS, but no robust change in gene expression was observed in vivo without the synergistic loss of the chromatin remodeler ISWI  . The sigAs Chd1 modulates PolII release from promoters, we wondered whether this ATP-remodeling complex alleviates PolII stalling. Therefore, we calculated the stalling index in the presence of wild-type and mutant Chd1. We defined the stalling index as the ratio of total PolII density at the promoter (−100 to +300 bp) relative to the density of PolII Ser2phos within the gene body (+1 to +5 kb) . We founTo confirm that the main function of Chd1 is at processive genes, Chd1 binding to stalled or processive genes was addressed. As Chd1 binding scales with PolII density , only thThe Chd1-binding profile was plotted for processive genes over the promoter region to understand how Chd1 might be functioning to enable promoter clearance at processive genes . WildtypDrosophila indicated that at the more processive M1BP bound genes, PolII accumulated in front of the well-positioned +1 nucleosome  are found on either H3 or H4, the retention of the H3:H4 tetramer during transcription has been proposed to have a role in the maintenance of chromatin states throughout the genome . This suPiggybac expression vector with the CAG promoter driving N-terminal FLAG-tagged expression of Chd1  and puromycin resistance. The K510R mutation was generated by site directed mutagenesis and verified by sequencing. MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% FBS, 1% penicillin-streptomycin at 37°C and puromycin (1.5 µg/ml). Knockdown of endogenous Chd1 was achieved using vectors as previously described ; Chd1 ; γ-Tubulin .ChIP was performed as previously described  with the2, 0.1% Triton X-100, 1 mM DTT, 20% Glycerol) and then processed as previously described. Input and CATCH-IT DNAs were amplified using a whole-genome amplification kit (Sigma-Aldrich), followed by labeling using Cy3 and Cy5 heptamers according to the Roche Nimblegen labeling protocol. Labeled samples were hybridized together to mouse 2.1-million probe promoter arrays .CATCH-IT was performed as described previously with the following modifications . Nuclei Data have been submitted to the Gene Expression Omnibus under accession number GSE52349 . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent -driven nucleosome turnover at gene promoters in vertebrate cells. Accordingly, Chd1 arises as a key regulator of RNAPII activity at the level of pausing and elongation. Given the widespread occurrence of RNAPII pausing in metazoans and the ever-increasing appreciation of RNAPII elongation control in regulation of gene expression, the current manuscript is deemed of high impact for the gene expression and chromatin fields.eLife basically as it is.The manuscript is beautifully written, the data is of high quality and generated with cutting-edge technology and the conclusions are fully supported by data. In my mind, the manuscript is meritorious for publication in My only minor concern is the creation of the RNAPII 'stalling index' in Reviewer2:The authors are experts in chromatin biology, and have developed several assays that enable different attributes of promoter chromatin to be analyzed. Here, they explore the impact of Chd1 remodeler on promoter chromatin. This issue has been addressed previously in ES cells at lower resolution, where promoter association was observed, with Chd1 loss leading to the acquisition of heterochromatin.The paper starts with the application of methods developed in the Henikoff lab to examine both Chd1 and RNA PolII in MEFs. This involves the separate examination of larger and smaller fragments derived from limited MNase digestion of crosslinked chromatin, following chromatin IP (ChIP). The larger fragments represent linkage of the factor to nucleosomes, whereas the smaller represent the factor's footprint. Support is provided that smaller fragments with PolII better link to the TSS.For Chd1, the authors examine both an epitope-tagged WT and a catalytic mutant. With both Chd1 derivatives, localization to the +1 nuc was observed, however the catalytic mutant showed additional occupancy of proximal downstream nucleosomes. This data was nicely done and described. The authors then explored the impact on nucleosome occupancy and positioning following expression of the dominant-negative Chd1 derivative. Here, they claim a loss in nucleosome occupancy without a change in positioning. I find the effect very small overall, even when PolII levels are considered, but much more clear in the case where the highest quintile of Chd1 occupancy is examined. The authors note a difference in impact here in MEFs on the +1 nuc, vs in budding yeast where no change is observed following Chd1 loss, which I found interesting. I also found the correlation of Chd1 with 'open' promoters interesting. The authors then apply an alternative shRNA approach to knock down Chd1, and observe similar (and even somewhat stronger) results.As prior approaches provide steady-state measurements, the authors then apply the CATCH-IT technique to explore dynamics. First, they show that turnover is high at locations flanking the TSS. The authors then explore the impact of expression of the dominant negative. The authors emphasize a significant difference at the locations flanking the TSS, and in the gene body. However, this is really confined to the nucleosome directly on, or adjacent to, the TSS; here it is clear and interesting, however, regarding the gene body, I found the difference with the dominant negative apparent, but the scale/magnitude quite small.The next section shows the promoter/TSS affect with the dominant negative scales with PolII to distinguish between increased recruitment vs lack of escape, relative distribution of elongating PolII, and  showed support for a role in promoter escape. Stalling index analysis show increases with the catalytic mutant and a preference for processive genes, which correlated with wt Chd1 occupancy. I found this data quite interesting. I found the data showing the peak of dominant negative Chd1 occupancy 50 bp inside the nucleosome very interesting, and consistent with current models of DNA translocation. Likewise, the peaks of PolII preceding nucleosomes in the early gene body supports the authors interesting claim of utilizing Chd1 activity to help facilitate PolII passage and reassembly behind the polymerase.Overall, I found the work well designed, executed and interpreted, and I strongly support its publication.Reviewer3:This manuscript investigates the role of Chd1 in transcription. Using MNase-seq, PolII ChIP-seq and CATCH-IT in MEF cells expressing WT or a dominant negative form of Chd1, the authors conclude that Chd1 is a factor required for the RNAP to overcome the barrier of the +1 nucleosome and relieve stalling of RNAP, whereas in the gene body it suppresses nucleosome turnover. There are some potentially good ideas in this manuscript, but the data do not significantly extend what we know from previous work and the data suffers from insufficiently rigorous analysis.These results are similar to work from a number of labs , showing that Chd1 localizes at promoters and within gene bodies where it facilitates transcription by altering nucleosome occupancy and turnover/histone replacement, especially at highly active genes. In particular, the data is reminiscent of Radman-Livaja et al. (PLOS Genetics 2012), where it is shown in yeast that loss of Chd1 activity leads to slower turnover of nucleosomes over the promoter and faster turnover in gene bodies and near 3' ends.S. cerevisiae showing that Chd1 is a promoter-specific chromatin remodeler, and work in S. pombe  showed that Chd1 localized particularly to promoter regions where it helped remove nucleosomes near the transcription start site to enable transcription. Thus, I don't see significant novelty in the majority of conclusions presented and don't find compelling the suggested connection between Chd1 activity at promoters and the metazoan-specific phenomenon of promoter stalling of RNAP. On this point, the hypothesis that PolII is being blocked in Chd1 mutant cells by nucleosomes despite the overall occupancy of the +1 nucleosome being lower is not clear to me. I can see how PolII could be blocked by an increase in nucleosome occupancy or by more static nucleosomes, but i.e. not what is indicated by this data. What the authors show is reduced +1 occupancy and reduced +1 turnover, which is most simply explained by nucleosomes being removed and not efficiently replaced. There is no clear data that sheds light on how this lower occupancy or failure to re-assemble nucleosomes might inhibit PolII.The authors emphasize the idea that Chd1 does not remodel promoter nucleosomes in yeast, but I don't understand why (they cite a Science paper from the Owen-Hughes lab that focused on nucleosome positioning). In fact that paper showed lower nucleosome occupancy near promoters in remodeling deficient cells, in total agreement with the current manuscript. Furthermore, the Kornberg lab  has done nice work in Major concerns:1) There are a number of cases where strong conclusions are drawn based on non-quantitative analysis of normalized group–average profiles. Group averages can be dramatically shifted by a small subset of genes and are not a reliable way to draw generalizable conclusions about a population. In cases where the authors wish to say that Chd1 mutation 'increases' or 'decreases' signal, statistical evaluations should be performed and described clearly in the methods section. When the authors wish to say that two signals 'correlate' with each other, correlation analysis should be performed and Pearson/Spearman R squared or similar values should be presented.Drosophila that shows normal Ser2-P PolII (active elongation form) levels in Chd1 mutants (Armstrong and Tamkun labs).2) It is really hard to interpret the data on PolII ChIP in cells expressing the catalytically dead Chd1 mutant without knowing what the effects of blocking Chd1 activity are on gene expression. The authors should at the very least select some example genes where loss of Chd1 increases the PolII stalling index and measure the nascent RNA levels at that gene by run-on or RT-PCR of intronic regions. The interpretation of the authors is that PolII is being 'blocked' at the promoter by the presence of more stable nucleosomes, but without showing that there is actually less active elongation within the gene bodies, this conclusion is very weakly supported, especially in light of work in Drosophila. In particular, Dave Gilmour's recent EMBO paper clearly shows that there are different classes of genes that differ in the role of the +1 nucleosome. Genes with the most paused PolII lack a significant +1 nucleosome signal and are paused by separate factors (GAGA and NELF) whereas less stably paused, more processive genes have a strong +1 nucleosome signal that causes more transient pausing. Do the authors find something significantly different in the mammalian system? Can they show by heatmap the relationship between the level of pausing and the occupancy level or turnover of the +1 nucleosome and analyze this quantitatively to demonstrate better that the +1 nucleosome might be a static barrier to mammalian PolII?3) It is very surprising that the authors don't mention any of the considerable work done on the relationship between PolII and the +1 nucleosome in  As you will gather from the full reviews included below, there was a clear disparity in the initial assessment of your manuscript. Whereas Reviewers 1 and 2 observed the various strengths of your work and found it appropriate for publication in eLife, Reviewer 3 pointed out that several key observations into the role of Chd1 in nucleosome remodeling at distinct genomic locations described in your manuscript had been previously established in the literature. This led Reviewers 1 and 2 to re-review your manuscript to determine to what degree the novel aspects in your work constituted a significant advance in the field. After this process, Reviewers 1 and 2 agreed that overall the paper ranks as very good and that there is new information in the manuscript.Upon further discussion, we would like to encourage a resubmission of a revised manuscript that not only thoroughly addresses the technical and data interpretation concerns raised by Reviewer 3, but also is rewritten in a way that better emphasizes what was known and what is novel in this paper about the role of Chd1 in nucleosome position and exchange. We also encourage addition of any new available data that would further advance our understanding of the molecular mechanism of action of Chd1.Thank you for taking the time to thoughtfully review our manuscript. We have addressed the concerns in the revision and have included a detailed description in response to the comments below.We believe that our initial submission did not emphasize the novel nature of our method for high resolution mapping of Chd1 and PolII. This technique is based upon the MNase digestion of crosslinked chromatin, limited sonication for quantitative ChIP recovery, and the sequencing of protected fragments. The development of the technique was essential for identification of Chd1 binding to the surface of nucleosomes, as previously implicated in vitro. In addition, this technique is highly adaptable, only requiring a simple modification to the regularly used ChIP-seq protocol and uses existing sequencing platforms. In the resubmission, we have provided further details of the method as a resource for the scientific community.As detailed below, we have rewritten the manuscript to address previous work on the role of Chd1 in yeast. In brief, from the point of view of this study, yeast differ in two main ways: (1) yeast have the TSS embedded within the +1 nucleosome, whereas in metazoans the entry site for the +1 nucleosome is ∼50 bp downstream; (2) unlike the situation in yeast, the vast majority of metazoan genes exhibit high levels of PolII accumulation near the promoter. As such, we set out to investigate Chd1 function in mammals. Our motivation was spurred on by the observation that Chd1 is required for metazoan specific phenomena – maintenance of pluripotency and reprogramming, for which we provide possible mechanistic insights. Also, understanding the regulation of nucleosome turnover in mammals is vital, for example with the recent observation that the widely used anti-cancer drug doxorubicin increases turnover, consistent with the tumor suppressor role of mammalian Chd1.We find a number of unique changes to chromatin that are not found in yeast: (1) the +1 nucleosome occupancy is decreased in the absence of Chd1 activity; (2) Chd1 primarily drives histone turnover at the promoter, compared to the relatively weak effect seen in the gene body. Work in yeast has not yielded a clear link between changes in chromatin and transcription upon loss of Chd1. Here we show that Chd1 occupancy and activity is linked to transcription, with Chd1 responsible for essentially all of PolII-dependent histone turnover at the promoter and removes the barrier to PolII promoter escape.Reviewer 1:My only minor concern is the creation of the RNAPII 'stalling index' in, which is basically equivalent to the RNAPII 'pausing index' used by many reports in the literature. There is a minor technical difference in how Skene et al. calculated their index  which I don't mind, but I don't think there is need to introduce 'stalling' here, I'll be happy with 'pausing'.The reason that we defined the term as ‘stalling index’ is for the subtle differences in the definition of stalled vs paused polymerases, as defined by John Lis . In brief, paused polymerases specifically are elongation competent, whereas the concept of a stalled polymerase is an inclusive term for all polymerase density . Our metric makes no assumption concerning the elongation potential of the polymerase and therefore we favor the term ‘stalling index.’Reviewer 2:The authors are experts in chromatin biology, and have developed several assays that enable different attributes of promoter chromatin to be analyzed. Here, they explore the impact of Chd1 remodeler on promoter chromatin. This issue has been addressed previously in ES cells at lower resolution, where promoter association was observed, with Chd1 loss leading to the acquisition of heterochromatin.The paper starts with the application of methods developed in the Henikoff lab to examine both Chd1 and RNA PolII in MEFs. This involves the separate examination of larger and smaller fragments derived from limited MNase digestion of crosslinked chromatin, following chromatin IP (ChIP). The larger fragments represent linkage of the factor to nucleosomes, whereas the smaller represent the factor's footprint. Support is provided that smaller fragments with PolII better link to the TSS.As mentioned above, the work presented here actually uses a novel technique that was specifically developed to address the distribution of Chd1. Typically our lab has favored native ChIP conditions. However, in mammalian cells we found that under native ChIP conditions only 14% of Chd1; 10% of PolII and ∼25% of histone H3 could be extracted, raising concerns that perhaps native ChIP would only investigate a specific genomic compartment of Chd1 binding. In comparison, sonication is typically used to both fragment chromatin and solubilize proteins in crosslinking ChIP, which results in low resolution and variable recovery. Here, we present the novel combination of formaldehyde crosslinking, MNase digestion to yield protected fragments and limited sonication to solubilize near 100% of Chd1 and chromatin. Through this technique we are able to generate high resolution data of Chd1 and PolII distributions. This was vital to observe the site of action of Chd1 on nucleosomes, as had been previously indicated in vitro (PMID: 16086025 and 16518397) and the stalled PolII in front of nucleosomes upon loss of Chd1 activity. This highly transferable technique has broad appeal to the scientific community, as it requires no special tool generation and uses current sequencing technology. The manuscript has been expanded to emphasize the novel nature of this technique, and For Chd1, the authors examine both an epitope-tagged WT and a catalytic mutant. With both Chd1 derivatives, localization to the +1 nuc was observed, however the catalytic mutant showed additional occupancy of proximal downstream nucleosomes. This data was nicely done and described. The authors then explored the impact on nucleosome occupancy and positioning following expression of the dominant-negative Chd1 derivative. Here, they claim a loss in nucleosome occupancy without a change in positioning. I find the effect very small overall, even when PolII levels are considered, but much more clear in the case where the highest quintile of Chd1 occupancy is examined. The authors note a difference in impact here in MEFs on the +1 nuc, versus in budding yeast where no change is observed following Chd1 loss, which I found interesting.In the revision, we now emphasize this difference, especially as it implies a fundamental difference between yeast and metazoans in how transcription proceeds through chromatin at the promoter.I also found the correlation of Chd1 with 'open' promoters interesting. The authors then apply an alternative shRNA approach to knock down Chd1, and observe similar (and even somewhat stronger) results.As prior approaches provide steady-state measurements, the authors then apply the CATCH-IT technique to explore dynamics. First, they show that turnover is high at locations flanking the TSS. The authors then explore the impact of expression of the dominant negative. The authors emphasize a significant difference at the locations flanking the TSS, and in the gene body. However, this is really confined to the nucleosome directly on, or adjacent to, the TSS; here it is clear and interesting, however, regarding the gene body, I found the difference with the dominant negative apparent, but the scale/magnitude quite small.We agree that the gene body turnover difference is small in absolute magnitude, which emphasizes that the biggest effect by far of mammalian Chd1 is at promoter proximal nucleosomes. This is fundamentally different from what is seen in yeast, where suppression of turnover in the gene body appears to be the primary role of yChd1 . We then extend this observation of mammalian Chd1 driving turnover and show that is responsible for essentially all of PolII directed nucleosome turnover, indicating its fundamental role in transcription through promoter proximal chromatin. The text has been modified to emphasize this novel role of Chd1, not evident from studies in yeast.Reviewer 3:This manuscript investigates the role of Chd1 in transcription. Using MNase-seq, PolII ChIP-seq and CATCH-IT in MEF cells expressing WT or a dominant negative form of Chd1, the authors conclude that Chd1 is a factor required for the RNAP to overcome the barrier of the +1 nucleosome and relieve stalling of RNAP, whereas in the gene body it suppresses nucleosome turnover. There are some potentially good ideas in this manuscript, but the data do not significantly extend what we know from previous work and the data suffers from insufficiently rigorous analysis.These results are similar to work from a number of labs , showing that Chd1 localizes at promoters and within gene bodies where it facilitates transcription by altering nucleosome occupancy and turnover/histone replacement, especially at highly active genes. In particular, the data is reminiscent of Radman-Livaja et al. (PLOS Genetics 2012), where it is shown in yeast that loss of Chd1 activity leads to slower turnover of nucleosomes over the promoter and faster turnover in gene bodies and near 3' ends.We are not disputing the importance of work that has been done with regards to Chd1 function in yeast. There are, however, key differences between our results in mammals and yeast, which likely reflects differences in promoter function and chromatin architecture. In addition, our work presents the novel finding that Chd1 functions to overcome the nucleosomal barrier to transcription, which has not been previously shown.In yeast, studies have focused on Chd1 suppressing histone turnover in the gene body , which we also observed in mammals, and further show that Chd1 recruitment to the gene body closely follows elongating PolII density. There are, however, fundamental differences in both transcription and chromatin context at the promoter between mammals and yeast. First, in yeast the transcription start site is embedded within the +1 nucleosome (PMID: 22258509), implying that the +1 nucleosome must be mobilized prior to pre-initiation complex formation. In contrast, in mammals the TSS is embedded within the nucleosome depleted region and the +1 nucleosome entry site found on average ∼50 bp downstream. Second, in mammals the vast majority of genes display PolII accumulation at the promoter region , whereas in yeast the PolII density is more evenly distributed along the length of the gene . This suggests a fundamental difference in the mechanism of PolII transit through the promoter proximal nucleosomes, which is supported by the identification of specific role of mammalian Chd1 at the promoter in driving turnover. In contrast to yeast where the magnitude of change in turnover at the promoter is approximately the same as in the gene body , the absence of mammalian Chd1 activity leads to a much greater reduction in promoter histone turnover. We present the novel finding that mammalian Chd1 is responsible for almost all PolII dependent turnover at the promoter and loss of this activity results in further accumulation of PolII at the promoter, a metazoan specific phenomenon. The previous yeast studies have not identified a consequence for transcription upon the loss of turnover at the promoter in the absence of Chd1. As such, our study identifies novel roles of Chd1 in terms of transcription related chromatin changes and places Chd1 as vital in the manipulation of metazoan specific chromatin structure.The text has been altered to include the yeast studies describing the role of Chd1 in turnover and emphasize that mammalian Chd1 is primarily functioning to drive turnover at the promoter in contrast to yeast (Results section entitled “Chd1 has opposing roles in regulating nucleosome turnover at the promoter and the gene body”).The authors emphasize the idea that Chd1 does not remodel promoter nucleosomes in yeast, but I don't understand why (they cite a Science paper from the Owen-Hughes lab that focused on nucleosome positioning). In fact that paper showed lower nucleosome occupancy near promoters in remodeling deficient cells, in total agreement with the current manuscript.This study  has done nice work in S. cerevisiae showing that Chd1 is a promoter-specific chromatin remodeler, and work in S. pombe  showed that Chd1 localized particularly to promoter regions where it helped remove nucleosomes near the transcription start site to enable transcription.These studies did not to identify a genome-wide link between Chd1 dependent chromatin changes and the changes in transcription. Here we have identified a unique role of mammalian Chd1 in regulating promoter nucleosome nuclear architecture in order to facilitate PolII promoter escape. These yeast studies have been acknowledged and discussed (In the Results section entitled “Chd1 activity is required for efficient promoter escape by PolII”).PHO5 promoter and indicated that Chd1 remodeling activity removed the +1 nucleosome in vitro . This is in contrast to the Owen-Hughes genome-wide analysis referred to above indicating no role for Chd1 in remodeling the +1 nucleosome (PMID: 21940898). This raises the possibility that Chd1 has a unique role at the PHO5 promoter. Additionally, the Kornberg study admits that in vivo PHO5 activation was robust in the chd1Δ strain, but required synergistic loss of ISWI to compromise gene activation. This raises the possibility of a separable function of Chd1 remodeling the yeast +1 nucleosome and ISWI-dependent transcription. In contrast, here we find absence of Chd1 activity alone has a genome-wide effect on metazoan-specific chromatin architecture and provide evidence for its vital role in overcoming the chromatin barrier to promoter escape via a novel high resolution mapping strategy.The Ehrensberger study (PMID: 21646535) investigated the chromatin structure at the S.pombe (PMID: 17510629) indicated that mutant strains displayed increased nucleosome density at Chd1-bound promoters, the opposite of what we observe here. This may reflect the unusually short 7 bp average linker length of S.pombe, which is very different from that of mammals, which is ∼50 bp. Moreover, the resolution (a single 500 bp Eurogentec microarray probe per promoter) was too low to draw distinctions between promoters and flanking nucleosomes, and only half of Chd1 bound promoters showed a change. The authors went on to look for changes in steady state RNA levels and found that 580 genes were upregulated and 238 genes were downregulated, suggesting that most genes were activated by the absence of Chd1. There was statistically insignificant overlap between the genes that showed increased nucleosome occupancy at the promoter upon loss of Chd1 and the expression changes . Therefore, it is important to separate steady state occupancy measurements from turnover. We find that a unique role of mammalian Chd1 is to drive PolII-dependent nucleosome turnover at the promoter, indicating the presence of more static nucleosomes at the promoter region in the absence of Chd1 activity. In the absence of Chd1 activity, we find that the barrier to PolII promoter escape is increased consistent with a more static chromatin barrier. The text has been altered to clarify this point (third paragraph of the Discussion).The reviewer presumes that nucleosome occupancy is correlated with turnover. However, work in yeast has shown that the highest turnover is at the promoter despite low nucleosome occupancy (PMID: 17347438). In Majorconcerns:1) There are a number of cases where strong conclusions are drawn based on non-quantitative analysis of normalized group-average profiles. Group averages can be dramatically shifted by a small subset of genes and are not a reliable way to draw generalizable conclusions about a population. In cases where the authors wish to say that Chd1 mutation 'increases' or 'decreases' signal, statistical evaluations should be performed and described clearly in the methods section. When the authors wish to say that two signals 'correlate' with each other, correlation analysis should be performed and Pearson/Spearman R squared or similar values should be presented.Statistical analysis has been performed and details included in the corresponding figure legends.2) It is really hard to interpret the data on PolII ChIP in cells expressing the catalytically dead Chd1 mutant without knowing what the effects of blocking Chd1 activity are on gene expression. The authors should at the very least select some example genes where loss of Chd1 increases the PolII stalling index and measure the nascent RNA levels at that gene by run-on or RT-PCR of intronic regions.We are interested in events occurring during transcription, whereas analysis of nascent chains can be complicated by co-transcriptional processing events. Therefore analyzing events that occur at the level of PolII are more relevant to the Chd1-related processes that we are studying here rather than analyzing the RNA produced downstream, especially as Chd1 has been proposed to interact with splicing factors (PMID: 18042460). In any case there is a close association between GRO-seq and PolII-Ser2-phos ChIP (PMID: 23062713), so nascent chain analysis is unlikely to provide any further insight. Therefore we favor our approach to measure ongoing transcription by genome-wide mapping of elongating PolII. Moreover we show that this reduction in elongating PolII density is more pronounced at genes with higher levels of Chd1 occupancy and is concomitant with an accumulation of PolII at the promoter, likely indicating a direct effect.The interpretation of the authors is that PolII is being 'blocked' at the promoter by the presence of more stable nucleosomes, but without showing that there is actually less active elongation within the gene bodies, this conclusion is very weakly supported, especially in light of work in Drosophila that shows normal Ser2-P PolII (active elongation form) levels in Chd1 mutants (Armstrong and Tamkun labs).Drosophila polytene chromosomes ”. Therefore, our observation of a decrease in genome-wide elongating PolII is not at all inconsistent with the results of this study.This previous observation of no change in Ser2-P PolII was based upon immunofluorescence of 3) It is very surprising that the authors don't mention any of the considerable work done on the relationship between PolII and the +1 nucleosome in Drosophila. In particular, Dave Gilmour's recent EMBO paper clearly shows that there are different classes of genes that differ in the role of the +1 nucleosome. Genes with the most paused PolII lack a significant +1 nucleosome signal and are paused by separate factors (GAGA and NELF) whereas less stably paused, more processive genes have a strong +1 nucleosome signal that causes more transient pausing. Do the authors find something significantly different in the mammalian system? Can they show by heatmap the relationship between the level of pausing and the occupancy level or turnover of the +1 nucleosome and analyze this quantitatively to demonstrate better that the +1 nucleosome might be a static barrier tomammalian PolII?Here we are investigating nucleosome dependent effects on PolII transit. NELF controls promoter proximal pausing, which occurs ∼30 bp downstream of the TSS, before the +1 nucleosome is encountered. We find that chromatin remodeler Chd1 preferentially operates on highly processive genes, which are independent of NELF regulation, to remove the nucleosome barrier to PolII. We have now altered the manuscript to include this work (last paragraph of the Results). We have also analyzed the data and find a similar situation in mammals, with processive genes having higher promoter proximal nucleosome occupancy and further show that the +1 nucleosome is more dynamic at processive genes . This su


Patients with persistent symptoms and/or villous atrophy despite strict adherence to a gluten-free diet (GFD) have non-responsive celiac disease (NRCD). A subset of these patients has refractory celiac disease (RCD), yet some NRCD patients may simply be reacting to gluten cross-contamination. Here we describe the effects of a 3-6 month diet of whole, unprocessed foods, termed the Gluten Contamination Elimination Diet (GCED), on NRCD. We aim to demonstrate that this diet reclassifies the majority of patients thought to have RCD type 1 (RCD1).We reviewed the records of all GFD-adherent NRCD patients cared for in our celiac center from 2005-2011 who were documented to have started the GCED. Response to the GCED was defined as being asymptomatic after the diet, with normal villous architecture on repeat biopsy, if performed.Prior to the GCED, all patients were interviewed by an experienced dietitian and no sources of hidden gluten ingestion were identified. 17 patients completed the GCED; 15 were female (88%). Median age at start of the GCED was 42 years (range 6-73). Fourteen patients (82%) responded to the GCED. Six patients met criteria for RCD prior to the GCED; 5 (83%) were asymptomatic after the GCED and no longer meet RCD criteria. Of the 14 patients who responded to the GCED, 11 (79%) successfully returned to a traditional GFD without resurgence of symptoms.The GCED may be an effective therapeutic option for GFD-adherent NRCD patients. Response to this diet identifies a subgroup of patients, previously classified as RCD1, that is not truly refractory to dietary treatment. Preventing an inaccurate diagnosis of RCD1 avoids immunotherapy. Most patients are able to return to a traditional GFD without return of symptoms. Celiac disease (CD) is an immune-mediated small intestinal enteropathy triggered by the ingestion of gluten in the genetically susceptible, with prevalence in the United States of nearly 1% ,3. The cBy far the most common cause of non-response is failure to adhere to the prescribed GFD, either voluntarily or unintentionally, thus highlighting the importance of a comprehensive dietary assessment ,8. AdditThe University of Maryland Center for Celiac Research in Baltimore, MD is a tertiary referral center for celiac disease and gluten-related disorders. As a celiac specialty clinic, many persistently symptomatic celiac patients are referred to our center for further evaluation and management of presumed RCD. Our initial evaluation of an NRCD patient begins with a comprehensive assessment of adherence to the diet by an expert dietitian, with focus not only on intentional noncompliance but also on determining the patient’s awareness regarding hidden sources of gluten to include communion wafers, medications and additives ,18,19. Wand/or villous atrophy despite being on a gluten free diet for ≥12 months. Prior to being considered for the GCED, dietary compliance was assessed by an experienced dietitian with expertise in celiac disease; patients must have been strictly adherent to the GFD, with no identifiable source of continued gluten exposure. Histological changes on duodenal biopsies were staged according to the Marsh Oberhuber classification [and villous atrophy (Marsh 3), despite a strict GFD for ≥12 months. If IEL immunophenotyping was performed on biopsy specimens, either by CD3/CD8 immunostaining or by T-cell receptor gene rearrangement analysis by polymerase chain reaction (PCR), RCD is further subdivided into RCD1 or RCD2 depending on the absence or presence, respectively, of a monoclonal IEL population. Celiac serology (IgA antibodies to tissue transglutaminase (tTG) and/or anti-endomysial antibodies (EMA)) is defined as negative, weak positive or high using the cut-offs provided by the ELISA kit manufacturer. Response to the GCED is defined as being asymptomatic after the diet, with normal villous architecture (Marsh 0-2) on repeat biopsy, if performed. Although celiac serology is drawn prior to initiation of the GCED and upon follow-up, the presence or absence of celiac auto-antibodies is not used in the definition for NRCD or RCD, nor is normalization of celiac serology, if applicable, used as a criterion for response to the GCED.This study was approved by the Institutional Review Board of the University of Maryland, Baltimore (IRB), and was conducted in accordance with the ethical standards and regulatory requirements of the IRB. We define NRCD as biopsy-proven CD with persistence or relapse of symptoms fication . RCD is The GCED, as shown in Table We reviewed the charts of all patients cared for in our center from 2005 through 2011 who were instructed on the GCED and met above criteria for NRCD patients on a well-documented strict GFD. The list of patients receiving dietary instruction was drawn from the celiac dietitian database. Patients who did not follow-up in clinic or otherwise communicate with our clinic after diet education were excluded as data were unavailable, to include whether or not the diet was initiated. Compliance was determined by patient report and dietitian interview; declaration of stopping the diet before the prescribed minimum of 3 months is considered noncompliance.Of the 1,288 patients with CD seen by our center from 2005 to 2011, a total of 29 (2.3%) patients were instructed on the GCED and met criteria for diet-adherent NRCD. Eight of these had no further communication with our clinic (lost to follow-up). The remaining 21 patients were documented to have started the GCED, with 4 of them admitting to cessation of the GCED and return to a traditional GFD prior to 3 months (noncompliant). Of the 17 compliant patients, 15 were female (88%). The median age of this group at initial diagnosis of CD was 30 years (range 1.6 - 52). Median age at initiation of the GCED was 42 years (range 6 – 73); 5 patients were <21 years of age. 14 (82%) patients had primary NRCD, with no improvement since starting the GFD, on average, 3 years prior to the GCED (range 1.1 - 4.6). 3 (18%) had secondary NRCD, with previous remission of disease but subsequent relapse, on average, 2 years prior to the GCED (range 1 – 3). Prior to starting the GCED, all but one patient (94%) were symptomatic, with the asymptomatic patient initiating the GCED due to high serology and persistent villous atrophy. Of the symptomatic, the most common symptoms were diarrhea (50%), followed by fatigue (31%) and abdominal pain (31%). The demographic details and clinical symptoms are shown in Table Response rate of the GCED was 82%, with 14 of the 17 compliant patients responding to the diet. Results of the GCED in terms of symptom resolution, histology and celiac serology are discussed below. Table Prior to starting the GCED, 16 of the 17 compliant patients (94%) were symptomatic and all but 3 patients became asymptomatic during the GCED, giving a symptom response rate of 81% (13/16). Six patients met the criteria for RCD prior to initiation of the GCED, with persistent symptoms and Marsh 3 histology. Five out of these 6 (83%) had full resolution of their symptoms after the GCED and no longer meet criteria for RCD.All patients had villous atrophy (Marsh 3) on histology at the time of their initial CD diagnosis. Twelve of the 17 compliant patients (71%) had repeat duodenal biopsies taken before initiation of the GCED. Seven patients demonstrated Marsh 3a/b histology 2.5 months, on average, prior to the GCED (range 0 – 5). Of these, patients 5 and 9 demonstrated complete mucosal healing on repeat endoscopy 5-6 months after starting the GCED; patient 15 had a normal follow-up video capsule endoscopy. Patient 17 did not respond symptomatically; follow-up video capsule endoscopy demonstrated villous atrophy and the patient was started on corticosteroids for RCD. Two patients demonstrated Marsh 2 histology <1 month prior to the GCED; patient 11 was re-biopsied 1 month after the GCED and demonstrated normal (Marsh 0) histology. Three patients demonstrated Marsh 0–1 histology prior to the GCED. There were two patients without pre-GCED biopsies that underwent endoscopy after completion of the GCED; patient 7 had responded to the diet symptomatically and demonstrated histologic remission (Marsh 1); patient 10 did not respond symptomatically to the diet and had persistent villous atrophy on histology, with IEL polyclonality, prompting corticosteroid treatment for RCD1.All patients had elevated tTG celiac serology at ini-tial histologically-proven CD diagnosis. All compliant patients had celiac serology drawn prior to the GCED (average <2 months prior) and after initiation of the GCED (average <6 months). In all but one patient, tTG IgA was followed; EMA IgA was followed in a patient with type 1 diabetes since false positive tTG titers have been reported in subjects affected by type 1 diabetes . ThirteeOf the 14 patients who responded to the diet, 11 (79%) successfully returned to their previous traditional GFD without resurgence of symptoms or elevated serology. The mean duration of the GCED among this group was 4.3 months (range 3 – 6 months) and mean duration of documented follow-up after initiation of the GCED was 20 months (range 1 – 62 months). Of the remaining 3 patients, patient 3 has had recurrence of his symptoms  after attempting to return to a traditional GFD on at least 3 separate occasions and has elected to stay on the GCED indefinitely. Patient 6 is currently back on the GCED due to a recurrence of hypertransaminasemia and high celiac serology after returning to the traditional GFD. Patient 16 has been lost to follow-up and it is unknown whether or not the patient was able to successfully transition to a normal diet.Four patients had documented initiation of the GCED but admitted to stopping the diet prior to the initial 3 month prescription. Three (75%) of these patients started the GCED solely for symptoms; they were serology negative and previously demonstrated Marsh 0-1 histology on biopsy. One patient stopped the diet after 1.5 months due to complete resolution of diarrhea and bloating, with no resurgence of symptoms at 7-month follow-up. The other 2 patients stopped the diet 1.5-2.25 months after initiation due to no improvement in their symptoms . Normal-appearing mucosa was re-confirmed in both patients, one by push endoscopy (Marsh 0) and the other by video capsule endoscopy . The last patient was started on the GCED for high serology, symptoms of inadequate weight gain and Marsh 3b histology 1 month prior to initiation of the diet. The patient discontinued the diet after 3 weeks, feeling it was too restrictive, and continues to have symptoms and elevated serology.Our study describes the effects of a 3- 6 month diet of exclusively whole, unprocessed foods on clinical symptoms, mucosal pathology and celiac serology in patients with NRCD that were already properly following a strict GFD, as assessed by experienced dietitians with expertise in celiac disease. With the belief that the majority of such patients are continuing to react to miniscule amounts of gluten cross-contamination, the goal of this diet is to eliminate exposure to any possible source of gluten cross-contamination from packaged/processed foods, to include those labeled gluten-free. In this study, 82% of our study patients responded to the GCED, with resolution of symptoms and, when applicable, resolution of their prior celiac enteropathy. Most importantly, of the 6 study patients who met criteria for RCD prior to initiating the GCED, 5 (83%) responded to the diet; they did not, indeed, have RCD.The GCED is not an attempt to enforce dietary adherence on a patient with detected persistent gluten ingestion, rather it modifies an already strict GFD to remove the possibility of trace gluten cross-contamination. Most CD patients can safely tolerate approximately 10 mg gluten cross-contamination daily, or 500 grams of food containing 20 ppm of gluten. However, there is a tremendous degree of variability within this population and some patients may have worsening histological changes with very low daily gluten exposure -24. The There are a number of recent articles discussing the evaluation of NRCD, with a focus on the approach to finding other underlying etiologies, such as inadvertent gluten ingestion or microscopic colitis, that may explain persistent symptoms ,26,27. Iwithout an apparent source of continued gluten exposure undergo further medical evaluation and consideration for the GCED.Most of the patients referred to our celiac center for persistent symptoms, elevated serology, or enteropathy have unintentional dietary gluten secondary to a lack of proper dietary education. Therefore, our initial investigation begins with a comprehensive interview and evaluation by an experienced dietitian; only those patients not symptomatically and histologically respond to the diet should be diagnosed with RCD1 and treated appropriately [After ensuring dietary compliance, the primary focus of an NRCD evaluation should be to rule-out the presence of RCD2, as this tends to follow an aggressive clinical course, with a high mortality rate primarily due to the development of intestinal lymphoma. Early recognition of RCD2 allows timely and more aggressive therapy, and encourages the use of diagnostic modalities such as video capsule endoscopy and CT enterography in order to exclude malignancy . We recopriately ,28.Persistent positive tTG IgA serology cannot be used to differentiate RCD from those without sustained mucosal damage, nor can it be used to differentiate RCD subtypes . FurtherAs Marsh 2 histology has, by definition, normal villi, we do not include this classification among those patients with potential RCD, despite the persistence of pathologic features. However, in those symptomatic CD patients with elevated serology, neither do we consider Marsh 2 lesions to be indicative of disease remission. 2 patients in this study had Marsh 2 histology prior to the GCED; both improved serologically and symptomatically and one underwent repeat biopsy with subsequent normal (Marsh 0) histology. For this reason we recommend that those patients with Marsh 2 histology undergo the GCED prior to any further work-up/evaluation. Our study also included two symptomatic patients with positive celiac serology yet Marsh 1 histology prior to initiation of the GCED; both of whom had subsequent resolution of symptoms and improved serology. Marsh 1 lesions would be consistent with disease remission, suggesting that these patients’ diarrheal symptoms were secondary to irritable bowel syndrome (IBS) or an underlying etiology other than gluten contamination. However, while we recognize that any diet modification carries with it some degree of placebo effect, the correlation of symptom improvement with a change in celiac serology is intriguing and the fact that both patients were subsequently able to return to their traditional GFD further argues for a non-placebo response. It should also be noted that one patient’s endoscopic report documented visualization of signs of villous atrophy. This raises the possibility of patchy villous recovery and subsequent missed villous lesions by biopsy ,35. GiveEighty-eight percent of our patients in this retrospective study were female. Although, as with other autoimmune diseases, there is a female predominance in CD, we were surprised that only 2 men were treated with the GCED since its development in 2005. As this study evaluated only those patients with NRCD who were treated with the GCED, we should not assume that this represents the entirety of our GFD-diet adherent NRCD population. However a previous study, after discounting those patients with inadvertent gluten ingestion, had an 81% female predominance in their NRCD population, supporting just such an assumption . LikewisOwing to the retrospective study design, a major limitation of our study is the absence of repeat biopsies in some of our study patients, with 5 patients not undergoing endoscopy prior to the GCED and only 5 patients being biopsied after completing the GCED. Whereas the definition of RCD mandates persistent symptoms and, thus, an asymptomatic patient would no longer meet criteria for RCD, documentation of mucosal healing in those patients who responded clinically and serologically would strengthen the study results. Moreover, a disease-specific celiac symptom index would have aided in reliably assessing symptom resolution. Although compliance with the GCED was assessed by dietitian interview, we must consider the reality that strictly adhering to such a limited diet is difficult and likely more costly than a standard strict GFD given the focus on fresh, non-processed foods; these factors may affect adherence disclosure. Our study is also limited by lack of information on NRCD patients that were not started on the GCED, and we must recognize the possibility of a selection bias toward expected response to the GCED among our study subjects. Additionally, 8 patients who met NRCD criteria and were instructed on the GCED by our nutritionist never followed-up with our clinic and were therefore not included in our study. As it is our dietitian’s experience that GCED patients typically contact her periodically for clarification on diet, these patients likely did not start the diet. However, some of these patients may have complied with the diet and simply followed-up with their primary GI physician; as such, we may be missing useful data. Lastly, as it was our decision to include all NRCD patients who initiated the GCED, symptomatic patients with both negative serology and healed mucosa were not excluded; 75% of the noncompliant patients met this description, as well as one of the compliant patients. These patients essentially had little evidence to suggest that removal of trace gluten contamination would significantly affect their symptoms.The GCED may be an effective therapeutic option for NRCD patients that have already failed a well-documented strict GFD and may aid in differentiating those patients reacting to trace amounts of gluten contamination from those who truly have RCD1. By avoiding an inaccurate diagnosis of RCD1, patients are able to avoid corticosteroids or immunotherapy. The expense and potential adverse health effects of this type of therapy make a dietary solution, aimed at the underlying etiology, particularly attractive. Moreover, a successful trial of the GCED in those patients with a high likelihood of having gluten cross-contamination, for instance those with high celiac serology, would avoid further unnecessary medical work-up. Most patients who respond to a 3-6 month course of the GCED are subsequently able to return to a traditional GFD without return of symptoms. Multi-center studies implementing the GCED in subjects affected by NRCD despite strict diet-adherence should be performed to confirm our data and, if confirmed, to re-evaluate the actual incidence of RCD1 as it may currently be over-estimated.CD: Celiac disease; RCD: Refractory celiac disease; NRCD: Non-responsive celiac disease; GFD: Gluten-free diet; GCED: Gluten contamination elimination diet.The authors have no competing interests to declare.The authors’ responsibilities were as follows—JH: designed the study, conducted the research, and was the primary author for the manuscript; PC: contributed to the conception and design of the study and created the subject database; EL, MM and AF : contributed to the conception and design of the study, interpretation of results, and assisted in writing the manuscript. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/13/40/prepub