Brain and cancer associated binding domain mutations provide insight into CTCF's relationship with chromatin and its ability to act as a chromatin organizer.

Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects.

Molecular models of bidirectional promoter regulation.

Approximately 11% of human genes are transcribed by a bidirectional promoter (BDP), defined as two genes with <1 kb between their transcription start sites. Despite their evolutionary conservation and enrichment for housekeeping genes and oncogenes, the regulatory role of BDPs remains unclear. BDPs have been suggested to facilitate gene coregulation and/or decrease expression noise. This review discusses these potential regulatory functions through the context of six prospective underlying mechanistic models: a single nucleosome free region, shared transcription factor/regulator binding, cooperative negative supercoiling, bimodal histone marks, joint activation by enhancer(s), and RNA-mediated recruitment of regulators. These molecular mechanisms may act independently and/or cooperatively to facilitate the coregulation and/or decreased expression noise predicted of BDPs.

An autoinhibitory switch of the LSD1 disordered region controls enhancer silencing.

Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements. Hydrogen-deuterium exchange shows that the LSD1 IDR interconverts between transient open and closed conformational states, the latter of which inhibits partitioning of the protein's structured domains with TF condensates. This autoinhibitory switch controls leukemic differentiation by modulating repression of active cis-regulatory elements bound by LSD1 and master hematopoietic TFs. Together, these studies unveil alternative mechanisms by which disordered regions and their dynamic crosstalk with structured regions can shape coregulator-TF interactions to control cis-regulatory landscapes and cell fate.

Dissection of a CTCF topological boundary uncovers principles of enhancer-oncogene regulation.

Enhancer-gene communication is dependent on topologically associating domains (TADs) and boundaries enforced by the CCCTC-binding factor (CTCF) insulator, but the underlying structures and mechanisms remain controversial. Here, we investigate a boundary that typically insulates fibroblast growth factor (FGF) oncogenes but is disrupted by DNA hypermethylation in gastrointestinal stromal tumors (GISTs). The boundary contains an array of CTCF sites that enforce adjacent TADs, one containing FGF genes and the other containing ANO1 and its putative enhancers, which are specifically active in GIST and its likely cell of origin. We show that coordinate disruption of four CTCF motifs in the boundary fuses the adjacent TADs, allows the ANO1 enhancer to contact FGF3, and causes its robust induction. High-resolution micro-C maps reveal specific contact between transcription initiation sites in the ANO1 enhancer and FGF3 promoter that quantitatively scales with FGF3 induction such that modest changes in contact frequency result in strong changes in expression, consistent with a causal relationship.

Brain and cancer associated binding domain mutations provide insight into CTCF's relationship with chromatin and its ability to act as a chromatin organizer.

Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects.

Integrative PTEN Enhancer Discovery Reveals a New Model of Enhancer Organization.

Enhancers possess both structural elements mediating promoter looping and functional elements mediating gene expression. Traditional models of enhancer-mediated gene regulation imply genomic overlap or immediate adjacency of these elements. We test this model by combining densely-tiled CRISPRa screening with nucleosome-resolution Region Capture Micro-C topology analysis. Using this integrated approach, we comprehensively define the cis-regulatory landscape for the tumor suppressor PTEN, identifying and validating 10 distinct enhancers and defining their 3D spatial organization. Unexpectedly, we identify several long-range functional enhancers whose promoter proximity is facilitated by chromatin loop anchors several kilobases away, and demonstrate that accounting for this spatial separation improves the computational prediction of validated enhancers. Thus, we propose a new model of enhancer organization incorporating spatial separation of essential functional and structural components.

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging.

PARP1 contributes to genome architecture and DNA damage repair through its dynamic association with chromatin. PARP1 and PARP2 (PARP1/2) recognize damaged DNA and recruit the DNA repair machinery. Using single-molecule microscopy in live cells, we monitored the movement of PARP1/2 on undamaged and damaged chromatin. We identify two classes of freely diffusing PARP1/2 and two classes of bound PARP1/2. The majority (>60%) of PARP1/2 diffuse freely in both undamaged and damaged nuclei and in the presence of inhibitors of PARP1/2 used for cancer therapy (PARPi). Laser-induced DNA damage results in a small fraction of slowly diffusing PARP1 and PARP2 to become transiently bound. Treatment of cells with PARPi in the presence of DNA damage causes subtle changes in the dynamics of bound PARP1/2, but not the high levels of PARP1/2 trapping seen previously. Our results imply that next-generation PARPi could specifically target the small fraction of DNA-bound PARP1/2.

Hydroxylation of methylated CpG dinucleotides reverses stabilisation of DNA duplexes by cytosine 5-methylation.

Cytosine-5-methylation stabilises DNA duplexes and is associated with transcriptional repression; 5-methylcytosine undergoes hydroxylation to 5-hydroxymethylcytosine, a modification of unknown biological function. Spectroscopic and calorimetric analyses show that 5-hydroxymethylcytosine introduction reverses the stabilising effect of 5-methylcytosine, suggesting that in some contexts, 5-methylcytosine hydroxylation may, along with other factors, contribute to the alleviation of transcriptional repression.