Landscape of DNA Methylation on the Marsupial X.

DNA methylation plays a key role in maintaining transcriptional silence on the inactive X chromosome of eutherian mammals. Beyond eutherians, there are limited genome wide data on DNA methylation from other vertebrates. Previous studies of X borne genes in various marsupial models revealed no differential DNA methylation of promoters between the sexes, leading to the conclusion that CpG methylation plays no role in marsupial X-inactivation. Using reduced representation bisulfite sequencing, we generated male and female CpG methylation profiles in four representative vertebrates (mouse, gray short-tailed opossum, platypus, and chicken). A variety of DNA methylation patterns were observed. Platypus and chicken displayed no large-scale differential DNA methylation between the sexes on the autosomes or the sex chromosomes. As expected, a metagene analysis revealed hypermethylation at transcription start sites (TSS) of genes subject to X-inactivation in female mice. This contrasted with the opossum, in which metagene analysis did not detect differential DNA methylation between the sexes at TSSs of genes subject to X-inactivation. However, regions flanking TSSs of these genes were hypomethylated. Our data are the first to demonstrate that, for genes subject to X-inactivation in both eutherian and marsupial mammals, there is a consistent difference between DNA methylation levels at TSSs and immediate flanking regions, which we propose has a silencing effect in both groups.

Stability of gene expression and epigenetic profiles highlights the utility of patient-derived paediatric acute lymphoblastic leukaemia xenografts for investigating molecular mechanisms of drug resistance.

BACKGROUND: Patient-derived tumour xenografts are an attractive model for preclinical testing of anti-cancer drugs. Insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. As a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale DNA methylation and gene expression profiling on a panel of 10 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) tumours that were stratified by prednisolone response. RESULTS: We found high correlations in DNA methylation and gene expression profiles between matching primary and xenograft tumour samples with Pearson's correlation coefficients ranging between 0.85 and 0.98. In order to demonstrate the potential utility of epigenetic analyses in BCP-ALL xenografts, we identified DNA methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. Differential methylation of CAPS2, ARHGAP21, ARX and HOXB6 were confirmed by locus specific analysis. We identified 20 genes showing an inverse relationship between DNA methylation and gene expression in association with prednisolone response. Pathway analysis of these genes implicated apoptosis, cell signalling and cell structure networks in prednisolone responsiveness. CONCLUSIONS: The findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric BCP-ALL propagated in mouse xenograft models. Further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses.

Correction for 'artificial' electron disequilibrium due to cone-beam CT density errors: implications for on-line adaptive stereotactic body radiation therapy of lung.

Cone-beam computed tomography (CBCT) has rapidly become a clinically useful imaging modality for image-guided radiation therapy. Unfortunately, CBCT images of the thorax are susceptible to artefacts due to scattered photons, beam hardening, lag in data acquisition, and respiratory motion during a slow scan. These limitations cause dose errors when CBCT image data are used directly in dose computations for on-line, dose adaptive radiation therapy (DART). The purpose of this work is to assess the magnitude of errors in CBCT numbers (HU), and determine the resultant effects on derived tissue density and computed dose accuracy for stereotactic body radiation therapy (SBRT) of lung cancer. Planning CT (PCT) images of three lung patients were acquired using a Philips multi-slice helical CT simulator, while CBCT images were obtained with a Varian On-Board Imaging system. To account for erroneous CBCT data, three practical correction techniques were tested: (1) conversion of CBCT numbers to electron density using phantoms, (2) replacement of individual CBCT pixel values with bulk CT numbers, averaged from PCT images for tissue regions, and (3) limited replacement of CBCT lung pixels values (LCT) likely to produce artificial lateral electron disequilibrium. For each corrected CBCT data set, lung SBRT dose distributions were computed for a 6 MV volume modulated arc therapy (VMAT) technique within the Philips Pinnacle treatment planning system. The reference prescription dose was set such that 95% of the planning target volume (PTV) received at least 54 Gy (i.e. D95). Further, we used the relative depth dose factor as an a priori index to predict the effects of incorrect low tissue density on computed lung dose in regions of severe electron disequilibrium. CT number profiles from co-registered CBCT and PCT patient lung images revealed many reduced lung pixel values in CBCT data, with some pixels corresponding to vacuum (-1000 HU). Similarly, CBCT data in a plastic lung phantom were reduced by 200 HU compared with known CT number values. For the three patients, dose results using the CBCT number data registered with PCT showed a prescription dose reduction ranging from 4 to 13% (D95 = 47 Gy). Therefore, accurate determination of lung density, especially for very low lung density (<0.2 g cm(-3)) is essential, but difficult to achieve using the CBCT data. Applying corrective techniques (1) and (2) to CBCT patient data produced unacceptable dose differences. For one typical VMAT SBRT patient, the D95 for the corrected CBCT and BCT image-based plans differed by -4% (D95 = 52 Gy) and 9% (D95 = 59 Gy) compared to the co-registered PCT image-based plan. However, corrective technique (3) produced negligible dose differences comparing LCT and PCT image-based plans. With regard to implementing on-line DART, dose errors must be minimized because they affect re-optimization decisions, and prevent accurate accumulation of the dose distribution.

Experimental assessments of intrafractional prostate motion on sequential and simultaneous boost to a dominant intraprostatic lesion.

PURPOSE: To investigate experimentally the impact of intrafractional prostate motion on the delivered dose to a dominant intraprostatic lesion (DIL) using volumetric modulated arc therapy (VMAT) and intensity modulated radiation therapy (IMRT) with sequential and simultaneous boost. METHODS: A series of six IMRT and VMAT treatment plans were generated, evaluated, and compared for two patient CT scans with dissimilar anatomies. Plans were generated for the prostate with and without the DIL. Plans were delivered using a Varian CLINAC and 2D dose distributions were measured using mapcheck(TM)-mapphan(TM) system. The effect of the prostate intrafractional motion on the delivery of the plans was studied by delivering the plans to the mapcheck(TM)-mapphan(TM) system on a programmable motion platform. Prostate intrafractional motion was simulated based on six different motion patterns from the literature obtained on Calypso system (Calypso System, Calypso Medical, Seattle, WA, USA) in a clinical study that provided continuous, real-time localization, and monitoring of the prostate. Absolute dose differences and Gamma analysis were used to assess the quality of a total of 42 plans with motion and without motion. RESULTS: Dose escalation to the whole prostate from 76 to 86 Gy caused the rectum and bladder to exceed normal tissue tolerances in both patients. All the DIL boost plans satisfied the planning criteria and delivery quality assurance when motion was not present. For a single fraction, the motion pattern with large constant shift caused the largest dose delivery discrepancy with mean Gamma value (1.14-1.44) and the lowest plan passing percentage (18.9%-35.7%), while the motion pattern with continuous random changes during treatment had the least impact on dose delivery with mean Gamma value (0.33-0.55) and the highest passing percentage (81.9%-100%) for all the investigated plans. For dose escalation to DIL in the presence of intrafractional prostate motion, a significant difference was observed between the different motion patterns (p < 0.05), but no significant difference in the sensitivity to motion between the various plans was observed (p = 0.30). Based on Gamma analysis, treatment courses in which 15% of the fractions are dominated by severe motion proved to be significantly different from those dominated by random motion (p < 0.05). CONCLUSIONS: The impact of intrafractional prostate motion on dose delivery is sensitive to different motion patterns but not to different delivery techniques. Dose escalation to DIL using either sequential or simultaneous boost plans with 7 mm PTV margin is achievable in the presence of intrafractional prostate motion, even if the severe motion comprised 8.6% (3 out of the 35) treatment fractions.

Stress-sensitive neurosignalling in depression: an integrated network biology approach to candidate gene selection for genetic association analysis.

Genetic risk for depressive disorders is poorly understood despite consistent suggestions of a high heritable component. Most genetic studies have focused on risk associated with single variants, a strategy which has so far only yielded small (often non-replicable) risks for depressive disorders. In this paper we argue that more substantial risks are likely to emerge from genetic variants acting in synergy within and across larger neurobiological systems (polygenic risk factors). We show how knowledge of major integrated neurobiological systems provides a robust basis for defining and testing theoretically defensible polygenic risk factors. We do this by describing the architecture of the overall stress response. Maladaptation via impaired stress responsiveness is central to the aetiology of depression and anxiety and provides a framework for a systems biology approach to candidate gene selection. We propose principles for identifying genes and gene networks within the neurosystems involved in the stress response and for defining polygenic risk factors based on the neurobiology of stress-related behaviour. We conclude that knowledge of the neurobiology of the stress response system is likely to play a central role in future efforts to improve genetic prediction of depression and related disorders.

Intensity modulated radiotherapy of non-small-cell lung cancer incorporating SPECT ventilation imaging.

PURPOSE: The authors performed this retrospective study to investigate the impact of using ventilation scans obtained from single photon emission computed tomography (SPECT) in selecting beam directions in intensity modulated radiation therapy (IMRT) planning in lung cancer radiotherapy to spare dosimetrically well ventilated lung. METHODS: For ten consecutive stage III non-small-cell lung cancer patients, the authors obtained both ventilation/perfusion SPECT scans and four-dimensional CT scans for treatment planning purposes. Each ventilation scan was registered with the corresponding planning CT and ventilation volumes corresponding to either > or = 50% (vv50) or > or = 70% (vv70) of the maximum SPECT count were automatically segmented. For each patient, three IMRT plans were generated: One using nine equally spaced beams optimized according to nonfunctional lung based mean lung dose and lung v20; a second using nine equally spaced beams optimized to avoid vv50 and vv70; and a third plan using only three beams with gantry angles chosen based on minimum mean ventilated lung dose calculated for each conformal beam at every 10 degrees gantry angle avoiding vv50 and vv70. Resultant dose volume histogram indices were calculated for each plan and were compared with respect to calculated SPECT-based ventilation parameters in order to quantify the potential utility of ventilation SPECT in this setting. RESULTS: Two patient groups were identified based on (i) the overlap volume between PTV and vv50 and (ii) the average angular mean ventilated lung dose (AAMvLD). The first parameter quantifies the proximity of the PTV to well ventilated lung and the second parameter quantifies the degree of ventilation that surrounds the PTV. For group 1 patients, < or = 5% of the vv50 overlapped with the PTV. For group 2 patients, > 5% of the vv50 overlapped the PTV. Group 1 was further classified into subgroups 1A and 1B: For subgroup 1A, AAMvLD is >18 Gy, implying that the functional lung surrounds the PTV; for subgroup 1B, AAMvLD is <18 Gy, implying that the well ventilated lung does not completely surround PTV. For subgroup 1A, the plans generated using ventilated lung avoidance reduced dose to vv50 and vv70, with below tolerance dose to normal lung and acceptable coverage of the PTV. For subgroup 1B, the dose to the total lung and well ventilated lung are reduced with the beam direction optimization for the three-beam plan. For group 2, there was no significant dosimetric advantage of using SPECT-based ventilation information in IMRT plan optimization. CONCLUSIONS: In conclusion, it is feasible to use SPECT ventilation scans to optimize IMRT beam direction and, subsequently, to reduce dose to ventilated lung when overlap of the PTV and the ventilated lung is minimal and that the PTV is not surrounded by the ventilated lung. The potential benefit of ventilation SPECT scanning can be determined by preplanning assessment of overlap volumes and the AAMvLD.

A case-sibling assessment of the association between skin pigmentation and other vitamin D-related factors and type 1 diabetes mellitus.

Fair skin pigmentation has been associated with a higher risk of type 1 diabetes mellitus (T1DM). The aim is to compare children with T1DM directly to a sibling in relation to their skin pigmentation in sun-exposed and unexposed sites, past sun exposure and methylation of the VDR gene promoter. The sample consisted of children with T1DM attending a diabetes outpatient clinic and siblings (total n=42). Cutaneous melanin density was estimated using a spectrophotometer. Parental report on past sun exposure was obtained. DNA methylation analysis of the VDR gene promoter was conducted. Matched data analysis was performed comparing each case directly to their sibling. Cases were significantly more likely to have lighter skin pigmentation at the upper arm (AOR 0.69 [95% CI: 0.52, 0.90]; P=0.01). Low infant sun exposure was imprecisely associated with a two-fold increase in T1DM risk (AOR 2.43 [95% CI: 0.91, 6.51]; P=0.08 for under 1 h of winter sun exposure per leisure day). The VDR gene promoter was completely unmethylated in both cases and siblings. The previously demonstrated association between light skin pigmentation and T1DM risk was evident even in this comparison across sibling pairs. Further work on past UVR exposure and related factors such as skin pigmentation is required.

Imprinted CDKN1C is a tumor suppressor in rhabdoid tumor and activated by restoration of SMARCB1 and histone deacetylase inhibitors.

SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi), Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

Experimental measurements and Monte Carlo simulations for dosimetric evaluations of intrafraction motion for gated and ungated intensity modulated arc therapy deliveries.

Respiratory gated radiation therapy allows for a smaller margin expansion for the planning target volume (PTV) to account for respiratory induced motion and is emerging as a common method to treat lung and liver tumors. We investigated the dosimetric effect of free motion and gated delivery for intensity modulated arc therapy (IMAT) with experimental measurements and Monte Carlo simulations. The impact of PTV margin and duty cycle for gated delivery is studied with Monte Carlo simulations. A motion phantom is used for this study. Two sets of contours were drawn on the mid-inspiration CT scan of this motion phantom. For each set of contours, an IMAT plan to be delivered with constant dose rate was created. The plans were generated on a CT scan of the phantom in the static condition with 3 mm PTV margin and applied to the motion phantom under four conditions: static, full superior-inferior (SI) motion (A = 1 cm, T = 4 s) and gating conditions (25% and 50% duty cycles) with full SI motion. A 6 by 15 cm piece of radiographic film was placed in the sagittal plane of the phantom and then irradiated under all measurement conditions. Film calibration was performed with a step-wedge method to convert optical density to dose. Gated IMAT delivery was first validated in 2D by comparing static film with that from gating and full motion. A previously verified simulation tool for IMRT that takes the log files from the multileaf collimator (MLC) controller and the gating system were adapted to simulate the delivered IMAT treatment for full 3D dosimetric analysis. The IMAT simulations were validated against the 2D film measurements. The resultant IMAT simulations were evaluated with dose criteria, dose-volume histograms and 3D gamma analysis. We validated gated IMAT deliveries when we compared the static film with the one from gating using 25% duty cycle using 2D gamma analysis. Within experimental and setup uncertainties, film measurements agreed with their corresponding simulated plans using 2D gamma analysis. Finally, when planning with margins designed for gating with 25% duty cycle and applying 50% or no gating during treatment, the dose differences in D(min,) D(99%) and D(95%) of the clinical target volume can be up to 27 cGy, 20 cGy and 18 cGy, respectively, for a plan with 200 cGy prescription dose. We have experimentally delivered gated IMAT with constant dose rate to a motion phantom and assessed their accuracies with film dosimetry and Monte Carlo simulations. Film dosimetry demonstrated that 25% gating and static plans are within 2%, 2 mm. The Monte Carlo simulation method was employed to generate dose delivered in 3D to a motion phantom, and the dosimetric results were reported. Since our film measurements agreed well with Monte Carlo simulations, we can reliably use this simulation tool to further study the dosimetric effects of target motion and effectiveness of gating for IMAT deliveries.

Incorporating geometric ray tracing to generate initial conditions for intensity modulated arc therapy optimization.

PURPOSE AND BACKGROUND: Intensity modulated arc therapy (IMAT) is a rotational variant of Intensity modulated radiation therapy (IMRT) that is achieved by allowing the multileaf collimator (MLC) positions to vary as the gantry rotates around the patient. This work describes a method to generate an IMAT plan through the use of a fast ray tracing technique based on dosimetric and geometric information for setting initial MLC leaf positions prior to final IMAT optimization. METHODS AND MATERIALS: Three steps were used to generate an IMAT plan. The first step was to generate arcs based on anatomical contours. The second step was to generate ray importance factor (RIF) maps by ray tracing the dose distribution inside the planning target volume (PTV) to modify the MLC leaf positions of the anatomical arcs to reduce the maximum dose inside the PTV. The RIF maps were also segmented to create a new set of arcs to improve the dose to low dose voxels within the PTV. In the third step, the MLC leaf positions from all arcs were put through a leaf position optimization (LPO) algorithm and brought into a fast Monte Carlo dose calculation engine for a final dose calculation. The method was applied to two phantom cases, a clinical prostate case and the Radiological Physics Center (RPC)'s head and neck phantom. The authors assessed the plan improvements achieved by each step and compared plans with and without using RIF. They also compared the IMAT plan with an IMRT plan for the RPC phantom. RESULTS: All plans that incorporated RIF and LPO had lower objective function values than those that incorporated LPO only. The objective function value was reduced by about 15% after the generation of RIF arcs and 52% after generation of RIF arcs and leaf position optimization. The IMAT plan for the RPC phantom had similar dose coverage for PTV1 and PTV2 (the same dose volume histogram curves), however, slightly lower dose to the normal tissues compared to a six-field IMRT plan. CONCLUSION: The use of a ray importance factor can generate initial IMAT arcs efficiently for further MLC leaf position optimization to obtain more favorable IMAT plan.

Commissioning a fast Monte Carlo dose calculation algorithm for lung cancer treatment planning.

A commercial Monte Carlo simulation package, NXEGS 1.12 (NumeriX LLC, New York, NY), was commissioned for photon-beam dose calculations. The same sets of measured data from 6-MV and 18-MV beams were used to commission NXEGS and Pinnacle 6.2b (Philips Medical Systems, Andover, MA). Accuracy and efficiency were compared against the collapsed cone convolution algorithm implemented in Pinnacle 6.2b, together with BEAM simulation (BEAMnrc 2001: National Research Council of Canada, Ottawa, ON). We investigated a number of options in NXEGS: the accuracy of fast Monte Carlo, the re-implementation of EGS4, post-processing technique (dose de-noising algorithm), and dose calculation time. Dose distributions were calculated with NXEGS, Pinnacle, and BEAM in water, lung-slab, and air-cylinder phantoms and in a lung patient plan. We compared the dose distributions calculated by NXEGS, Pinnacle, and BEAM. In a selected region of interest (7725 voxels) in the lung phantom, all but 1 voxel had a gamma (3% and 3 mm thresholds) of 1 or less for the dose difference between the NXEGS re-implementation of EGS4 and BEAM, and 99% of the voxels had a gamma of 1 or less for the dose difference between NXEGS fast Monte Carlo and BEAM. Fast Monte Carlo with post-processing was up to 100 times faster than the NXEGS re-implementation of EGS4, while maintaining +/- 2% statistical uncertainty. With air inhomogeneities larger than 1 cm, post-processing preserves the dose perturbations from the air cylinder. When 3 or more beams were used, fast Monte Carlo with post-processing was comparable to or faster than Pinnacle 6.2b collapsed cone convolution.

Monte Carlo dose calculation of segmental IMRT delivery to a moving phantom using dynamic MLC and gating log files.

Respiratory gating is emerging as a tool to limit the effect of motion for liver and lung tumors. In order to study the impact of target motion and gated intensity modulated radiation therapy (IMRT) delivery, a computer program was developed to simulate segmental IMRT delivery to a moving phantom. Two distinct plans were delivered to a rigid-motion phantom with a film insert in place under four conditions: static, sinusoidal motion, gated sinusoidal motion with a duty cycle of 25% and gated sinusoidal motion with duty cycle of 50% under motion conditions of a typical patient (A = 1 cm, T = 4 s). The MLC controller log files and gating log files were retained to perform a retrospective Monte Carlo dose calculation of the plans. Comparison of the 2D planar dose distributions between simulation and measurement demonstrated that our technique had at least 94% of the points passing gamma criteria of 3% for dose difference and 3 mm as the distance to agreement. This note demonstrates that the use of dynamic multi-leaf collimator and respiratory monitoring system log files together with a fast Monte Carlo dose calculation algorithm is an accurate and efficient way to study the dosimetric effect of motion for gated or non-gated IMRT delivery on a rigidly-moving body.

Segmentation and leaf sequencing for intensity modulated arc therapy.

A common method in generating intensity modulated radiation therapy (IMRT) plans consists of a three step process: an optimized fluence intensity map (IM) for each beam is generated via inverse planning, this IM is then segmented into discrete levels, and finally, the segmented map is translated into a set of MLC apertures via a leaf sequencing algorithm. To date, limited work has been done on this approach as it pertains to intensity modulated arc therapy (IMAT), specifically in regards to the latter two steps. There are two determining factors that separate IMAT segmentation and leaf sequencing from their IMRT equivalents: (1) the intrinsic 3D nature of the intensity maps (standard 2D maps plus the angular component), and (2) that the dynamic multileaf collimator (MLC) constraints be met using a minimum number of arcs. In this work, we illustrate a technique to create an IMAT plan that replicates Tomotherapy deliveries by applying IMAT specific segmentation and leaf-sequencing algorithms to Tomotherapy output sinograms. We propose and compare two alternative segmentation techniques, a clustering method, and a bottom-up segmentation method (BUS). We also introduce a novel IMAT leaf-sequencing algorithm that explicitly takes leaf movement constraints into consideration. These algorithms were tested with 51 angular projections of the output leaf-open sinograms generated on the Hi-ART II treatment planning system (Tomotherapy Inc.). We present two geometric phantoms and 2 clinical scenarios as sample test cases. In each case 12 IMAT plans were created, ranging from 2 to 7 intensity levels. Half were generated using the BUS segmentation and half with the clustering method. We report on the number of arcs produced as well as differences between Tomotherapy output sinograms and segmented IMAT intensity maps. For each case one plan for each segmentation method is chosen for full Monte Carlo dose calculation (NumeriX LLC) and dose volume histograms (DVH) are calculated. In all cases, the BUS method outperformed the clustering, method. We recommend using the BUS algorithm and discuss potential improvements to the clustering algorithms.

Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis.

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L-/- germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L-/- meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

Brahma links the SWI/SNF chromatin-remodeling complex with MeCP2-dependent transcriptional silencing.

Transcriptional repression of methylated genes can be mediated by the methyl-CpG binding protein MeCP2. Here we show that human Brahma (Brm), a catalytic component of the SWI/SNF-related chromatin-remodeling complex, associates with MeCP2 in vivo and is functionally linked with repression. We used a number of different molecular approaches and chromatin immunoprecipitation strategies to show a unique cooperation between Brm, BAF57 and MeCP2. We show that Brm and MeCP2 assembly on chromatin occurs on methylated genes in cancer and the gene FMR1 in fragile X syndrome. These experimental findings identify a new role for SWI/SNF in gene repression by MeCP2.