Francois Le Dily

Nucleosome-Driven Transcription Factor Binding and Gene Regulation

Elucidating the global function of a transcription factor implies the identification of its target genes and genomic binding sites. The role of chromatin in this context is unclear, but the dominant view is that factors bind preferentially to nucleosome-depleted regions identified as DNaseI-hypersensitive sites (DHS). Here we show by ChIP, MNase, and DNaseI assays followed by deep sequencing that the progesterone receptor (PR) requires nucleosomes for optimal binding and function. In breast cancer cells treated with progestins, we identified 25,000 PR binding sites (PRbs). The majority of these sites encompassed several copies of the hexanucleotide TGTYCY, which is highly abundant in the genome. We found that functional PRbs accumulate around progesterone-induced genes, mainly in enhancers. Most of these sites overlap with DHS but exhibit high nucleosome occupancy. Progestin stimulation results in remodeling of these nucleosomes with displacement of histones H1 and H2A/H2B dimers. Our results strongly suggest that nucleosomes are crucial for PR binding and hormonal gene regulation.

CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer.

Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes

A close chromatin conformation precludes gene expression in eukaryotic cells. Genes activated by external cues have to overcome this repressive state by locally changing chromatin structure to a more open state. Although much is known about hormonal gene activation, how basal repression of regulated genes is targeted to the correct sites throughout the genome is not well understood. Here we report that in breast cancer cells, the unliganded progesterone receptor (PR) binds genomic sites and targets a repressive complex containing HP1γ (heterochromatin protein 1γ), LSD1 (lysine-specific demethylase 1), HDAC1/2, CoREST (corepressor for REST [RE1 {neuronal repressor element 1} silencing transcription factor]), KDM5B, and the RNA SRA (steroid receptor RNA activator) to 20% of hormone-inducible genes, keeping these genes silenced prior to hormone treatment. The complex is anchored via binding of HP1γ to H3K9me3 (histone H3 tails trimethylated on Lys 9). SRA interacts with PR, HP1γ, and LSD1, and its depletion compromises the loading of the repressive complex to target chromatin-promoting aberrant gene derepression. Upon hormonal treatment, the HP1γ-LSD1 complex is displaced from these constitutively poorly expressed genes as a result of rapid phosphorylation of histone H3 at Ser 10 mediated by MSK1, which is recruited to the target sites by the activated PR. Displacement of the repressive complex enables the loading of coactivators needed for chromatin remodeling and activation of this set of genes, including genes involved in apoptosis and cell proliferation. These results highlight the importance of the unliganded PR in hormonal regulation of breast cancer cells.

Two Chromatin Remodeling Activities Cooperate during Activation of Hormone Responsive Promoters

Steroid hormones regulate gene expression by interaction of their receptors with hormone responsive elements (HREs) and recruitment of kinases, chromatin remodeling complexes, and coregulators to their target promoters. Here we show that in breast cancer cells the BAF, but not the closely related PBAF complex, is required for progesterone induction of several target genes including MMTV, where it catalyzes localized displacement of histones H2A and H2B and subsequent NF1 binding. PCAF is also needed for induction of progesterone target genes and acetylates histone H3 at K14, an epigenetic mark that interacts with the BAF subunits by anchoring the complex to chromatin. In the absence of PCAF, full loading of target promoters with hormone receptors and BAF is precluded, and induction is compromised. Thus, activation of hormone-responsive promoters requires cooperation of at least two chromatin remodeling activities, BAF and PCAF.

ADP-ribose–derived nuclear ATP synthesis by NUDIX5 is required for chromatin remodeling

A nuclear power source in the cell DNA is packaged onto nucleosomes, the principal component of chromatin. This chromatin must be remodeled to allow gene transcription, DNA replication, and DNA repair machineries access to the enclosed DNA. Chromatin-remodeling complexes require high levels of cellular energy to do their job. Wright et al. show that the energy needed to remodel chromatin can be derived from a source, poly-ADP-ribose, in the cell nucleus, rather than by diffusion of ATP from mitochondria in the cytoplasm, the usual powerhouse of the cell. Poly-ADP-ribose is converted to ADP-ribose and then to ATP, which can be used to fuel chromatin remodeling within the nucleus. Science, this issue p. 1221 Energy needed to remodel chromatin to make DNA accessible can be generated in situ in the nucleus from ADP-ribose. Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation.

Nuclear factor 1 synergizes with progesterone receptor on the mouse mammary tumor virus promoter wrapped around a histone H3/H4 tetramer by facilitating access to the central hormone-responsive elements.

Steroid hormones induce transcription of their responsive genes by complex mechanisms including synergism between the hormone receptors and other transcription factors. On the mouse mammary tumor virus (MMTV) promoter progesterone induction is mediated by the reciprocal synergism between progesterone receptor (PR) and the ubiquitous transcription factor nuclear factor 1 (NF1). PR binding mediates ATP-dependent displacement of histone H2A and H2B, enabling NF1 access to its target site. In minichromosomes assembled in vitro NF1 binding facilitates access of PR to the hormone-responsive elements (HREs) by precluding reforming of the histone octamer, but the function of NF1 in living cells remains unclear. Here we show that depleting NF1 by small interfering RNAs or mutating the NF1-binding site significantly compromises transcription of the MMTV promoter. The central HREs 2 and 3 are not needed for ATP-dependent H2A/H2B displacement or NF1 binding but are critical for full PR binding and MMTV transactivation. We found that NF1 binding to the MMTV promoter on a H3/H4 histone tetramer particle exposes the central HREs and facilitates their binding by PR, suggesting a possible mechanism for the reciprocal synergism between PR and NF1.

On the demultiplexing of chromosome capture conformation data.

How to describe the multiple chromosome structures that underlie interactions among genome loci and how to quantify the occurrence of these structures in a cell population remain important challenges to solve, which can be addressed via a proper demultiplexing of chromosome capture conformation related data. Here, we first aim to review two main methodologies that have been proposed to tackle this problem: restrained‐based methods, in which the resulting chromosome structures stem from the multiple solutions of a distance satisfaction problem; and thermodynamic‐based methods, in which the structures stem from the simulation of polymer models. Next, we propose a novel demultiplexing method based on a matrix decomposition of contact maps. To this end, we extend the notion of topologically associated domains (TADs) by introducing that of statistical interaction domains (SIDs). SIDs can overlap and occur in a cell population at certain frequencies, and we propose a simple method to estimate these frequency values. As an application, we show that SIDs that measure 100 kb to tens of Mb long occur both frequently and specifically in the human genome.

TADs as modular and dynamic units for gene regulation by hormones

During cell differentiation epigenetic processes permit the establishment of a cell type specific transcriptome by limiting the fraction of the genome that will be expressed. Based upon steady‐state requirements and transcription factor expression, differentiated cells respond transiently to external cues by modulating the expression levels of subsets of genes. Increasing evidence demonstrates that the genome is organized non‐randomly in a hierarchy of structures within the nuclear space, where chromosome territories are segmented into Topologically Associating Domains (TADs) and sub‐domains. It remains poorly understood how this three‐dimensional organization of the genome participates in the acquisition of a cell‐specific program of gene expression. Furthermore, it is unknown whether this spatial framework influences the dynamic changes of gene expression that accompany alterations in the cell environment. In this review, we will discuss the impact of genome topology on the response of breast cancer cells to steroid hormones. We will cover steroid nuclear receptor mechanisms of action and discuss how topological organization of the genome, including segmentation into TADs, acts as a combinatorial platform to integrate signals whilst ultimately ensuring coordinate regulation of gene expression.

Hi-Cpipe: a pipeline for high-throughput chromosome capture

HiC-inspector is a toolkit for the analysis and visualization of data generated by high-throughput chromatin conformation capture (HiC). The analysis module comprises steps of data formatting, genome alignment, quality control and filtering, identification of genome-wide chromatin interactions, and statistics. The interactive browser enables visual inspection of interaction data generated and analysis results.

Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization

Two variant cohesin complexes containing SMC1, SMC3, RAD21 and either SA1 (also known as STAG1) or SA2 (also known as STAG2) are present in all cell types. We report here their genomic distribution and specific contributions to genome organization in human cells. Although both variants are found at CCCTC-binding factor (CTCF) sites, a distinct population of the SA2-containing cohesin complexes (hereafter referred to as cohesin-SA2) localize to enhancers lacking CTCF, are linked to tissue-specific transcription and cannot be replaced by the SA1-containing cohesin complex (cohesin-SA1) when SA2 is absent, a condition that has been observed in several tumors. Downregulation of each of these variants has different consequences for gene expression and genome architecture. Our results suggest that cohesin-SA1 preferentially contributes to the stabilization of topologically associating domain boundaries together with CTCF, whereas cohesin-SA2 promotes cell-type-specific contacts between enhancers and promoters independently of CTCF. Loss of cohesin-SA2 rewires local chromatin contacts and alters gene expression. These findings provide insights into how cohesin mediates chromosome folding and establish a novel framework to address the consequences of mutations in cohesin genes in cancer.Depletion, ChIP and Hi-C analyses of the genomic distribution of the two variant cohesin complexes in human cells reveal non-redundant functions and differential contributions to 3D genome organization.