The cohesin acetylation cycle controls chromatin loop length through a PDS5A brake mechanism

Cohesin structures the genome through the formation of chromatin loops and by holding together the sister chromatids. The acetylation of cohesin’s SMC3 subunit is a dynamic process that involves the acetyltransferase ESCO1 and deacetylase HDAC8. Here we show that this cohesin acetylation cycle controls the three-dimensional genome in human cells. ESCO1 restricts the length of chromatin loops, and of architectural stripes emanating from CTCF sites. HDAC8 conversely promotes the extension of such loops and stripes. This role in controlling loop length turns out to be distinct from the canonical role of cohesin acetylation that protects against WAPL-mediated DNA release. We reveal that acetylation controls the interaction of cohesin with PDS5A to restrict chromatin loop length. Our data support a model in which this PDS5A-bound state acts as a brake that enables the pausing and restart of loop enlargement. The cohesin acetylation cycle hereby provides punctuation in the process of genome folding

Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw

Aspects of Exercise before or after Bariatric Surgery: A Systematic Review

Background: Bariatric surgery has a considerable effect on weight loss. A positive relation of exercise and weight loss has been described before. However, the mode of exercise and its timing pre- or postoperatively or a combination remains unclear. Methods: A multi-database search was conducted. Identified articles were reviewed on description of exercise, timing around a bariatric intervention, and outcome. Methodological quality of the included studies was rated using the Physiotherapy Evidence Database scale. A Cohen's kappa score assessed the level of agreement. Outcome measurements were improvement of anthropometric and physical fitness variables, operation related complications, weight regain, and quality of life. Results: A total of 8 prospective studies were included. Four focused on training before and 4 on training after a bariatric procedure. Details of exercises varied from 45 min treadmill up to full descriptive programs. Supervision was frequently included. Significant improvement was encountered for biometric results physical fitness variables. Conclusion: In the majority of reports on exercising in a (future) bariatric population, positive effects on anthropometrics, cardiovascular risk factors and physical fitness were described. However, the results were not unanimous, with a wide range of exercise programs and perioperative timing, therefore hampering adequate practical guidance

Characterization and dynamics of pericentromere-associated domains in mice

Despite recent progress in genome topology knowledge, the role of repeats, which make up the majority of mammalian genomes, remains elusive. Satellite repeats are highly abundant sequences that cluster around centromeres, attract pericentromeric heterochromatin, and aggregate into nuclear chromocenters. These nuclear landmark structures are assumed to form a repressive compartment in the nucleus to which genes are recruited for silencing. We have designed a strategy for genome-wide identification of pericentromere-associated domains (PADs) in different mouse cell types. The ∼1000 PADs and non-PADs have similar chromatin states in embryonic stem cells, but during lineage commitment, chromocenters progressively associate with constitutively inactive genomic regions at the nuclear periphery. This suggests that PADs are not actively recruited to chromocenters, but that chromocenters are themselves attracted to inactive chromatin compartments. However, we also found that experimentally induced proximity of an active locus to chromocenters was sufficient to cause gene repression. Collectively, our data suggest that rather than driving nuclear organization, pericentromeric satellite repeats mostly co-segregate with inactive genomic regions into nuclear compartments where they can contribute to stable maintenance of the repressed status of proximal chromosomal regions

CTCF Binding Polarity Determines Chromatin Looping

CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure

Robust 4C-seq data analysis to screen for regulatory DNA interactions

Regulatory DNA elements can control the expression of distant genes via physical interactions. Here we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using chromosome conformation capture combined with high-throughput sequencing (4C-seq). Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation

The structural basis for cohesin-CTCF-anchored loops

Cohesin catalyses the folding of the genome into loops that are anchored by CTCF1. The molecular mechanism of how cohesin and CTCF structure the 3D genome has remained unclear. Here we show that a segment within the CTCF N terminus interacts with the SA2–SCC1 subunits of human cohesin. We report a crystal structure of SA2–SCC1 in complex with CTCF at a resolution of 2.7 Å, which reveals the molecular basis of the interaction. We demonstrate that this interaction is specifically required for CTCF-anchored loops and contributes to the positioning of cohesin at CTCF binding sites. A similar motif is present in a number of established and newly identified cohesin ligands, including the cohesin release factor WAPL2,3. Our data suggest that CTCF enables the formation of chromatin loops by protecting cohesin against loop release. These results provide fundamental insights into the molecular mechanism that enables the dynamic regulation of chromatin folding by cohesin and CTCF