Roni H. G. Wright

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.

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.

Hormone‐induced repression of genes requires BRG1‐mediated H1.2 deposition at target promoters

Eukaryotic gene regulation is associated with changes in chromatin compaction that modulate access to DNA regulatory sequences relevant for transcriptional activation or repression. Although much is known about the mechanism of chromatin remodeling in hormonal gene activation, how repression is accomplished is much less understood. Here we report that in breast cancer cells, ligand‐activated progesterone receptor (PR) is directly recruited to transcriptionally repressed genes involved in cell proliferation along with the kinases ERK1/2 and MSK1. PR recruits BRG1 associated with the HP1γ‐LSD1 complex repressor complex, which is further anchored via binding of HP1γ to the H3K9me3 signal deposited by SUV39H2. In contrast to what is observed during gene activation, only BRG1 and not the BAF complex is recruited to repressed promoters, likely due to local enrichment of the pioneer factor FOXA1. BRG1 participates in gene repression by interacting with H1.2, facilitating its deposition and stabilizing nucleosome positioning around the transcription start site. Our results uncover a mechanism of hormone‐dependent transcriptional repression and a novel role for BRG1 in progestin regulation of breast cancer cell growth.

Linker histones in hormonal gene regulation

In the present review, we summarize advances in our knowledge on the role of the histone H1 family of proteins in breast cancer cells, focusing on their response to progestins. Histone H1 plays a dual role in gene regulation by hormones, both as a structural component of chromatin and as a dynamic modulator of transcription. It contributes to hormonal regulation of the MMTV promoter by stabilizing a homogeneous nucleosome positioning, which reduces basal transcription whereas at the same time promoting progesterone receptor binding and nucleosome remodeling. These combined effects enhance hormone dependent gene transcription, which eventually requires H1 phosphorylation and displacement. Various isoforms of histone H1 have specific functions in differentiated breast cancer cells and compact nucleosomal arrays to different extents in vitro. Genome-wide studies show that histone H1 has a key role in chromatin dynamics of hormone regulated genes. A complex sequence of enzymatic events, including phosphorylation by CDK2, PARylation by PARP1 and the ATP-dependent activity of NURF, are required for H1 displacement and gene de-repression, as a prerequisite for further nucleosome remodeling. Similarly, during hormone-dependent gene repression a dedicated enzymatic mechanism controls H1 deposition at promoters by a complex containing HP1γ, LSD1 and BRG1, the ATPase of the BAF complex. Thus, a broader vision of the histone code should include histone H1, as the linker histone variants actively participate in the regulation of the chromatin structure. How modifications of the core histones tails affect H1 modifications and vice versa is one of the many questions that remains to be addressed to provide a more comprehensive view of the histone cross-talk mechanisms.

ADP-ribose derived Nuclear ATP is Required for Chromatin Remodeling and Hormonal Gene Regulation

Highlights – Hormonal gene regulation requires synthesis of PAR and its degradation to ADP-ribose by PARG – ADP-ribose is converted to ATP in the cell nuclei by hormone-activated NUDIX5/NUDT5 – Blocking nuclear ATP formation precludes hormone-induced chromatin remodeling, gene regulation and cell proliferation Summary Key nuclear processes in eukaryotes including DNA replication or repair and gene regulation require extensive chromatin remodeling catalyzed by energy consuming enzymes. How the energetic demands of such processes are ensured in response to rapid stimuli remains unclear. We have 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. Nuclear ATP synthesis requires the combined enzymatic activities of PARP1, PARG and NUDIX5/NUDT5. Although initiated via mitochondrial derived ATP, the nuclear source of ATP is essential for hormone induced chromatin remodeling, gene regulation and cell proliferation and may also participate in DNA repair. This novel pathway reveals exciting avenues of research for drug development.

PARty promoters: hormone-dependent gene regulation requires CDK2 activation of PARP1.

Comment on: Wright RH, et al. Genes Dev 2012; 26:1972-83.

Progesterone receptor maintains estrogen receptor gene expression by regulating DNA methylation in hormone-free breast cancer cells

Background How breast cancers respond to endocrine therapy strongly depends on the expression of the estrogen and progesterone receptors (ER and PR, respectively), with double-negative ER–/PR– breast cancers having worse clinical outcome than ER+/PR+ breast cancers. Although much is known about ERα gene (ESR1) regulation after hormonal stimulation, how it is regulated in the absence of hormones is not fully understood. We used ER+/PR+ positive breast cancer cells to investigate the role of PR in ESR1 gene regulation in the absence of hormones. Methods We compared the ESR1 gene expression and its methylation profile between PR-positive breast cancer cells, PR-depleted cells and PR-rescue cells by RT-qPCR and MeDIP-qPCR, respectively. Moreover, we analyzed the PR binding to ESR1 locus by chromatin immunoprecipitation method and, to methylated or unmethylated progesterone responsive elements by electrophoresis mobility shift assay. Results We show that PR binds to the low-methylated ESR1 promoter and maintains both gene expression and the DNA methylation profile of the ESR1 locus in hormone-deprived breast cancer cells. Depletion of PR reduces ESR1 expression, with a concomitant increase in gene promoter methylation. The high amount of DNA methylation in the ESR1 promoter of PR-depleted cells persists after the stable re-expression of PR and inhibits PR binding to this genomic region. Consequently, the rescue of PR expression in PR-depleted cells is insufficient to restore ESR1 expression. Consistent with these data, DNA methylation impedes PR binding to consensus progesterone responsive elements in vitro. Conclusion These findings help us understand the complex crosstalk between PR and ER, and suggest that the analysis of DNA methylation of ESR1 promoter in breast cancer cells can help to design the appropriate targeted therapies for different types of breast cancer patients

Insight into the machinery that oils chromatin dynamics

ABSTRACT The packaging of genetic information in form of chromatin within the nucleus provides cells with the ability to store and protect massive amounts of information within a compact space. Storing information within chromatin allows selective access to specific DNA sequences by regulating the various levels of chromatin structure from nucleosomes, to chromatin fibers, loops and topological associating domains (TADs) using mechanisms that are being progressively unravelled. However, a relatively unexplored aspect is the energetic cost of changing the chromatin configuration to gain access to DNA information. Among the enzymes responsible for regulating chromatin access are the ATP-dependent chromatin remodellers that act on nucleosomes and use the energy of ATP hydrolysis to make chromatin DNA more accessible. It is assumed that the ATP used by these enzymes is provided by the mitochondria or by cytoplasmic glycolysis. We hypothesize that though this may be the case for cells in steady state, when gene expression has to be globally reprogramed in response to externals signals or stress conditions, the cell directs energy production to the cell nucleus, where rapid chromatin reorganization is needed for cell survival. We discovered that in response to hormones a nuclear ATP synthesis mechanism is activated that utilizing ADP-ribose and pyrophosphate as substrates.1 This extra view aims to put this process within its historical context, to describe the enzymatic steps in detail, to propose a possible structure of the ATP synthesising enzyme, and to shed light on how this may link to other reactions within the cell providing a perspective for future lines of investigation.

DNA damage and gene transcription: accident or necessity?

The extent to which DNA repair machinery facilitates gene activation remains poorly appreciated. A new study published in Cell Research reports a novel function of H2AX, a substrate of ATM and known DNA damage marker, in transcriptional initiation.

Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells

Breast cancer prognosis and response to endocrine therapy strongly depends on the expression of the estrogen and progesterone receptors (ER and PR, respectively). Although much is known about ERα gene (ESR1) regulation after hormonal stimulation, how it is regulated in hormone-free condition is not fully understood. We used ER-/PR-positive breast cancer cells to investigate the role of PR in ESR1 regulation in the absence of hormones. We show that PR binds to the low-methylated ESR1 promoter and maintains both gene expression and DNA methylation of the ESR1 locus in hormone-deprived breast cancer cells. Depletion of PR reduces ESR1 expression, with a concomitant increase in gene promoter methylation. The high amount of methylation in the ESR1 promoter of PR-depleted cells persists after the stable re-expression of PR and inhibits PR binding to this genomic region. As a consequence, the rescue of PR expression in PR-depleted cells is insufficient to restore ESR1 expression. Consistently, DNA methylation impedes PR binding to consensus progesterone responsive elements. These findings contribute to understanding the complex crosstalk between PR and ER and suggest that the analysis of ESR1 promoter methylation in breast cancer cells can help to design more appropriate targeted therapies for breast cancer patients.