BRCA1 mutations attenuate super-enhancer function and chromatin looping in haploinsufficient human breast epithelial cells
BRCA1-associated breast cancer originates from luminal progenitor cells. BRCA1 functions in multiple biological processes, including double-strand break repair, replication stress suppression, transcriptional regulation, and chromatin reorganization. While non-malignant cells carrying cancer-predisposing BRCA1 mutations exhibit increased genomic instability, it remains unclear whether BRCA1 haploinsufficiency affects transcription and chromatin dynamics in breast epithelial cells.
H3K27ac-associated super-enhancers were compared in primary breast epithelial cells from BRCA1 mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+). Non-tumorigenic MCF10A breast epithelial cells with engineered BRCA1 haploinsufficiency were used to confirm the H3K27ac changes. The impact of BRCA1 mutations on enhancer function and enhancer-promoter looping was assessed in MCF10A cells.
Here, we show that primary mammary epithelial cells from women with BRCA1 mutations display significant loss of H3K27ac-associated super-enhancers. These BRCA1-dependent super-enhancers are enriched with binding motifs for the GATA family. Non-tumorigenic BRCA1mut/+ MCF10A cells recapitulate the H3K27ac loss. Attenuated histone mark and enhancer activity in these BRCA1mut/+ MCF10A cells can be partially restored with wild-type BRCA1. Furthermore, chromatin conformation analysis demonstrates impaired enhancer-promoter looping in BRCA1mut/+ MCF10A cells.
H3K27ac-associated super-enhancer loss is a previously unappreciated functional deficiency in ostensibly normal BRCA1 mutation-carrying breast epithelium. Our findings offer new mechanistic insights into BRCA1 mutation-associated transcriptional and epigenetic abnormality in breast epithelial cells and tissue/cell lineage-specific tumorigenesis.
KeywordsBRCA1 Transcription Super-enhancer Chromatin looping Epigenetics Breast epithelial cells
Chromosome conformation capture
Bromodomain and extraterminal
Bromodomain-containing protein 4
Chromatin immunoprecipitation with deep sequencing
Histone lysine 27 acetylation
Histone deacetylase complex
Human mammary epithelial cell
- Pol II
RNA polymerase II
Positive transcription elongation factor b
Transcription start site
Approximately 1 in 400 women in the USA carry germ-line BRCA1 mutation (BRCA1mut/+) [1, 2]. These BRCA1 mutation carriers have significantly higher risk of developing breast cancer compared to the general population, with an estimated cumulative risk of 65% by the age of 70 [3, 4]. While breast cancer screening could assist diagnosis at an early stage, it alone cannot reduce cancer risk . The only effective risk-reducing options for women with BRCA1 mutations are prophylactic mastectomy and oophorectomy, which can achieve 90% and 50% reduction in breast cancer risk, respectively [6, 7, 8, 9]. However, due to the adverse physical and psychological effects, many at-risk women opt not to undergo these surgeries [10, 11]. Understanding functional deficiency that occurs prior to clinically evident cancer in precancerous BRCA1mut/+ breast epithelium is an important step towards developing alternative preventive strategies with higher precision and fewer side effects.
Mammary gland epithelium is composed of two lineages: luminal cells that surround the central lumen, and basal cells that are located adjacent to mammary stroma . BRCA1 haploinsufficiency leads to a luminal progenitor population deficiency in luminal cell differentiation [13, 14, 15, 16]. Most BRCA1-associated breast tumors have a basal-like phenotype, with positive staining for the basal cell markers cytokeratin 5/6/14/17 and negative staining for the luminal cell markers estrogen receptor (ER) and progesterone receptor (PR) [17, 18, 19, 20]. Of note, the basal breast cancer subtype is associated with poor clinical outcome . However, BRCA1-associated basal-like breast tumors originate from luminal progenitor cells, namely, the cell of origin for BRCA1-associated tumors [13, 14, 16]. A major gap of knowledge in BRCA1-related cancer biology concerns the mechanism by which a single copy of BRCA1 mutant allele leads to luminal differentiation deficiency and eventually basal-like tumors.
BRCA1 is best known for maintenance of genomic integrity through its functions in repair of double-strand DNA breaks via homologous recombination (HR) [22, 23, 24], regulation of cell cycle checkpoints [25, 26], and suppression of DNA replication stress . When compared with their BRCA1+/+ counterparts, BRCA1mut/+ mammary epithelial cells function comparably in checkpoint regulation, yet exhibit haploinsufficiency in replication stress suppression and DNA repair [27, 28, 29, 30, 31]. While maintenance of genomic integrity is essential to BRCA1 tumor suppressor function, it alone does not easily explain the cell lineage-specific deficiency that occurs at early stages of tumorigenesis in BRCA1 mutation carriers. BRCA1 is also implicated in transcriptional regulation and high-order chromatin reorganization [25, 32, 33, 34, 35, 36, 37], processes that primarily dictate normal tissue development and cell differentiation. In support of this notion, multiple genome-wide studies show that BRCA1 preferentially binds to transcription start sites (TSSs) [38, 39]. Furthermore, our recent mouse genetic studies provide evidence for a functional crosstalk between BRCA1 and a bona fide transcription factor that regulates mammary luminal progenitor cell expansion and BRCA1-associated tumorigenesis [15, 40]. However, it remains unclear whether BRCA1mut/+ breast epithelial cells are haploinsufficient in regulation of transcription and chromatin dynamics.
Acetylated histones destabilize nucleosomes, increase chromatin accessibility for transcription factor binding, and ultimately facilitate gene expression [41, 42]. In particular, histone lysine 27 acetylation (H3K27ac) serves as a surrogate mark for active transcriptional enhancers . Super-enhancers, which are large clusters of transcriptional enhancers, are bound by high levels of master regulatory transcription factors and co-factors [44, 45]. A high concentration of transcription factor binding renders rapid response of the corresponding target genes to various developmental cues [44, 46]. Super-enhancers, which are highly cell-type specific and enriched for H3K27ac, drive expression of genes that have essential roles in cell fate determination . Notably, dysfunctional super-enhancers have been causally linked to pathogenesis including cancer [44, 45, 47, 48, 49, 50, 51, 52]. Of note, BRCA1 interacts with CREB-binding protein (CBP) and p300, two structurally related histone acetyltransferases (HAT) that acetylate histones including H3K27 . In addition, BRCA1 is found to interact with components of the histone deacetylase complex (HDAC) . However, a potential role of BRCA1 in regulation of super-enhancer functions has not been investigated.
Here, we conducted whole-genome H3K27ac profiling of primary breast epithelial cells from BRCA1 mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+). Bioinformatics analysis indicates that heterozygous cancer-predisposing BRCA1 mutation (BRCA1mut/+) dampens super-enhancer marks in primary human mammary epithelial cells (HMECs), in particular at those super-enhancers with GATA transcription factor binding. The effect of BRCA1 mutations on super-enhancers was further corroborated using established non-tumorigenic breast epithelial cells engineered with a single copy of BRCA1 mutant allele (BRCA1mut/+). Mechanistically, reduced H3K27ac levels in BRCA1mut/+ cells lead to impaired enhancer-promoter looping and decreased enhancer activity. Our work uncovers a previously unappreciated function of BRCA1 in super-enhancer regulation. The functional haploinsufficiency likely contributes to the cell lineage switch observed in early stages of BRCA1-associated breast tumorigenesis.
Breast tissue cohorts
Cancer-free breast tissues were procured from women either undergoing cosmetic reduction mammoplasty or prophylactic mastectomy, following protocols approved by the Institutional Review Board at the University of Texas Health Science Center at San Antonio. All donors signed written consent forms authorizing the use of the specimens.
Primary epithelial cell isolation from human breast tissue
Fresh human breast tissue was processed as previously described . In brief, tissue was digested in digestion buffer (DMEM/F-12 supplemented with 5% FBS, 0.1% BSA, 10 ng/mL epidermal growth factor, 10 ng/mL cholera toxin, 5 μg/mL insulin, 0.5 mg/mL hydrocortisone, 300 U/mL collagenase, and 100 U/mL hyaluronidase) on a 37 °C shaker overnight. Epithelium-enriched population was collected by centrifugation at 100 g for 3 min. Pellet was treated with 0.8% ammonium chloride to lyse red blood cells, followed by digestion with 0.05% trypsin-EDTA at 37 °C for 3 min. Cells were washed with washing buffer (HBSS supplemented with 2% FBS) and treated with dispase buffer (5 mg/mL dispase supplemented with 0.1 mg/mL DNase I) at 37 °C for 3 min. Single cells were obtained by passing through a 40-μm strainer.
Chromatin immunoprecipitation (ChIP)
For H3K27ac/BRD4/CTCF ChIP, single cells were crosslinked with 1% formaldehyde at room temperature for 10 min, followed by incubation with 125 mM glycine for an additional 5 min. For MED1/BRCA1 ChIP, cells were crosslinked with 2 mM of disuccinimidyl glutarate (Thermo Fisher Scientific; 20593) at room temperature for 45 min, followed by further crosslinking with formaldehyde as described above. All following steps were carried out in buffers containing protease inhibitors in 4 °C until elution. Cells were pelleted by centrifugation at 1000g for 5 min, washed with PBS twice, then lysed in lysis buffer (5 mM HEPES, pH 7.9, 85 mM KCl, 0.5% Triton X-100) for 10 min. Nuclei were pelleted by centrifugation at 1600 g for 5 min and lysed in nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). Chromosomal DNA was sonicated using a Bioruptor Pico to obtain < 300-bp fragments. Ten percent of sonicated DNA was saved as input, and the rest was incubated with various antibodies overnight (H3K27ac: Abcam; ab4729. BRD4: Abcam; ab128874. CTCF: MilliporeSigma; 07-729. MED1: Bethyl Laboratories, Inc.; A300-793A. BRCA1: Bethyl Laboratories, Inc.; A300-000A). Dynabeads Protein A or G (Thermo Fisher Scientific; 10002D or 10003D) was added the following day and incubated for additional 4 h before washing. Washing was performed twice in TE sarcosyl buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.2% sarcosyl), twice in TSE1 buffer (150 mM sodium chloride, 20 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), twice in TSE2 buffer (500 mM sodium chloride, 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1% SDS, 0.1% Triton X-100), twice in TSE3 buffer (250 mM lithium chloride, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% sodium deoxycholate, 1% NP-40), and twice in TE buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA). DNA was subsequently eluted from Dynabeads, reverse-crosslinked, and ethanol-precipitated. Locus-specific ChIP was assessed by PCR using primers as shown in Additional file 1: Table S2.
For BRD4-H3K27ac ChIP-re-ChIP, samples were processed as described above prior to washing. BRD4-DNA-bound beads were washed three times in re-ChIP washing buffer (2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% NP40) and twice in TE buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA). Samples were eluted in re-ChIP elution buffer (2% SDS, 15 mM DTT in TE buffer) by incubation at 37 °C for 30 min. After diluting 20 times with dilution buffer (16.7 mM Tris-HCl, pH 8.0, 0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 50 μg of BSA), samples were incubated with the re-ChIP antibody overnight, then processed as ChIP samples using the method described above.
Library preparation and sequencing
H3K27ac chromatin immunoprecipitation with deep sequencing (ChIP-seq) libraries were constructed using a MicroPlex Library Preparation Kit (Diagenode; C05010011) following the manufacturer’s guide. After a total of 10 cycles of PCR amplification, libraries were purified using Agencourt AMPure XP System (Beckman Coulter; A63880). Quality and quantity of the libraries were measured by a Qubit dsDNA HS Assay Kit (Life Technologies; Q32851) using a Bioanalyser 2100. Libraries with different index sequences were pooled together and then sequenced with a single-end 50-bp module using an Illumina Hiseq 3000 system. De-multiplexing was performed by CASAVA to generate FASTQ files for each sample. Between 38 and 92 million unique mapped reads were obtained for each sample.
Bioinformatics analysis of ChIP-seq
H3K27ac ChIP-seq was aligned to the human genome by BWA , and only unique mapped reads were saved. BELT , a bin-based peak calling algorism that applies a statistical method to control false discovery rate (FDR), was used to call peaks. Super-enhancers were identified using ROSE [45, 46]. Briefly, H3K27ac ChIP-seq peaks within 12.5 kb of one another were stitched together as enhancer clusters, then ranked and plotted based on the H3K27ac ChIP-seq signal. Stitched enhancer clusters that pass the inflection point in the distribution were designated as super-enhancers. HOMER  program was used for prediction of transcription factor binding sites. H3K27ac peaks located within super-enhancers were pooled together for motif search. Each super-enhancer was assigned a gene name based on closest proximity. ToppGene was used for Gene Ontology analysis .
MCF10A with wild-type BRCA1 or heterozygous BRCA1 mutations were previously reported [29, 30] and cultured in DMEM/F12 (Thermo Fisher Scientific; 11330) supplemented with 5% of horse serum (Thermo Fisher Scientific; 16050), 20 ng/mL EGF (Gibco; PHG0311), 0.5 mg/mL hydrocortisone (Sigma; H0888), 100 ng/mL cholera toxin (Sigma; C8052), 10 μg/mL insulin (Sigma, I1882), and 1× penicillin-streptomycin (Thermo Fisher Scientific; 15070). Two days prior to the experiments, cells were trypsinized and 1.2 million cells were seeded in each 10-cm dish (MilliporeSigma; CLS3262).
Chromatin conformation analysis (3C)
3C was performed following an established protocol with minor changes [57, 58]. In brief, MCF10A cells were trypsinized and counted. Ten million cells were used for each 3C condition. Cells were crosslinked with 1% formaldehyde at room temperature for 10 min, followed by 125 mM glycine at room temperature for 5 min. Cells were pelleted by centrifugation at 600 g at 4 °C for 5 min and re-suspended in pre-chilled lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% NP-40, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin, and 1 mM PMSF). Samples were incubated on ice for 15 min and passed through a 21-G needle five times. Nuclei were pelleted by centrifugation at 2200g at 4 °C for 5 min, washed twice with NEB buffer 2.1, and re-suspended in NEB buffer 2.1. SDS was added to nuclei at a final concentration of 0.1%. Samples were incubated on a 65 °C shaker for 10 min then on ice immediately. Triton X-100 was added to quench SDS at a final concentration of 1%. Samples were incubated with 400 U of HindIII (New England Biolabs; R3104L) at 37 °C with rotation overnight. SDS was added to the samples the following day at a final concentration of 1.6%. Samples were incubated on a 65 °C shaker for 30 min and then transferred into 15-mL tubes with pre-chilled ligation buffer (1% Triton X-100, 0.8 mg BSA, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM DTT, and 2 mM ATP). After incubation with 300 U of T4 DNA ligase (Thermo Fisher Scientific; EL0011) at 16 °C for 4 h, samples were treated with 0.5 mg of proteinase K at 65 °C for 4 h, followed by an additional 0.5 mg of proteinase K treatment at 65 °C overnight. DNA was phenol-chloroform extracted the following day, diluted with distilled water, and ethanol-precipitated. Samples were treated with RNase A at 37 °C for 2 h, followed by phenol-chloroform extraction and ethanol precipitation. DNA was dissolved in TE at 4 °C overnight. Serially diluted 3C products were analyzed by PCR to determine linear range. 3C libraries within the linear range were analyzed by PCR using primers specific for the restriction fragments of interest. GAPDH was used for loading normalization. 3C primers are listed in the Additional file 1: Table S2.
BRCA1 mut/+ HMECs are associated with reduced super-enhancer mark
BRCA1 haploinsufficient MCF10A cells recapitulate H3K27ac changes in primary BRCA1 mut/+ HMECs
To corroborate the findings from primary HMECs, we used MCF10A cells that were genetically engineered to harbor a single allele of cancer-causing BRCA1 mutation [29, 30]. MCF10A represents an immortalized yet non-tumorigenic human breast epithelial cell line with near normal diploidy. When cultured with extracellular matrix, MCF10A cells form acinar structures that recapitulate many aspects of mammary architecture in vivo [64, 65]. Previously published work has shown that BRCA1mut/+ MCF10A cells are prone to genomic instability, thus mimicking HMECs of BRCA1 mutation carriers [29, 30].
Reduced H3K27ac level in BRCA1 haploinsufficient cells attenuates action of enhancer-binding proteins and transcription of target genes
We also sought to determine whether BRCA1 haploinsufficiency affects chromatin association of other enhancer-binding proteins at the aforementioned loci. MED1 is a subunit of the transcription coactivator Mediator complex that serves as a bridge to physically connect enhancers with their corresponding promoters and to transduce signals from various transcription factors to RNA polymerase II . MED1 chromatin binding at the BRCA1 mutation-affected super-enhancers showed a trend of decrease, albeit statistically insignificant, in BRCA1185delAG/+ MCF10A clones versus the WT control (Fig. 4c). We also assessed chromatin binding of CCCTC-binding factor (CTCF), which acts to shield undesired interactions between enhancers and promoters [73, 74]. Consistent with public datasets that indicate two CTCF binding peaks at the SOD2 super-enhancer (Additional file 3: Figure S3A), we found that CTCF ChIP signals at these sites have similar intensity in WT control and BRCA1185delAG/+cells (Fig. 4d). Global CTCF levels were also similar in WT and mutant clones (Additional file 3: Figure S6). In accordance with reduced H3K27ac and BRD4 binding at these two super-enhancer loci, mRNA levels of TNFAIP3 and SOD2 were significantly diminished in BRCA1185delAG/+ MCF10A clones (Fig. 4e). Taken together, our data clearly suggest that haploinsufficient BRCA1 mutation selectively impairs chromatin binding of enhancer-binding proteins and transcription of their downstream target genes.
Impaired enhancer-promoter looping in BRCA1 haploinsufficient cells
Combining studies of clinical samples and gene editing-generated isogenic cell lines, our work clearly demonstrates that a single copy of cancer-predisposing BRCA1 mutation reduces super-enhancer mark and enhancer function in transcriptional activation. The causality of BRCA1 haploinsufficiency and super-enhancer dysfunction is corroborated by partial rescuing of the phenotype with ectopic wild-type BRCA1. Collectively, our findings lend support to the notion that heterozygous BRCA1 mutations are haploinsufficient for transcriptional regulation in non-tumorigenic breast epithelial cells prior to clinically evident cancer appearance.
BRCA1-associated breast tumors originate from luminal progenitor cells, yet they eventually become basal-like [13, 14, 16]. Deficient luminal cell maturation represents one of the earliest hallmarks of BRCA1 mutation-carrying breast epithelium [14, 15]. Our data indicate that super-enhancers that are preferentially lost in BRCA1mut/+ HMECs are significantly enriched for GATA binding sites. Among the members of the evolutionally conserved GATA transcription factor family , GATA3 is known for its critical role in regulating luminal cell fate in the mammary gland [60, 61]. Notably, genetic ablation of mouse Gata3 causes expansion of luminal progenitor cells and deficiency in luminal differentiation, which bears striking resemblance to BRCA1-deficient mammary epithelium [14, 15, 60]. Of note, it was reported that BRCA1 and GATA3 physically interact with each other to regulate gene expression . Therefore, it is conceivable that BRCA1 promotes luminal differentiation by facilitating GATA3 transcriptional activity at the corresponding super-enhancers. We surmise that in breast epithelium of BRCA1 mutation carriers, BRCA1 haploinsufficiency could dampen GATA3 action in promoting luminal differentiation, which in turn drives the luminal-to-basal transition observed at early stages of BRCA1-associated breast tumorigenesis.
While the biochemical basis for BRCA1 function in enhancer-promoter looping remains to be elucidated, several possible mechanisms are worth considering. First, BRCA1 could reinforce enhancer-promoter looping by recruiting HATs such as p300 and thus increasing H3K27ac density . In a second scenario, BRCA1 is known to interact with RNA polymerase II (Pol II) and Pol II-pausing factor NELF-B/COBRA1 [33, 34]. In addition, BRD4 participates in regulation of transcription elongation [82, 83, 84]. In this regard, BRCA1 could strengthen enhancer-promoter looping through its interactions with factors involved in regulation of Pol II dynamics at the promoter-proximal region. In yet another alternative model, the potent ubiquitin E3 ligase activity of BRCA1/BARD1 heterodimeric complex has recently been implicated in histone H2A ubiquitination  and estrogen metabolism-related transcriptional regulation . It is therefore conceivable that BRCA1/BARD1 E3 ligase-mediated chromatin modification could impact enhancer-promoter looping . These possible mechanisms are not mutually exclusive, and further studies are warranted to shed more mechanistic light on three-dimensional chromatin reorganization in BRCA1 mutation-carrying breast epithelium.
Our gene ontology analyses indicate that genes proximal to BRCA1-associated super-enhancers are enriched with those involved in cellular responses to various physiological cues including inflammation and stress. In particular, those involved in NF-κB and retinoic acid responses were identified in a previous study by Gardini et al. using an in vitro BRCA1 knock-down system in MCF10A cells . Moreover, deregulated progesterone signaling [93, 94] and persistently active NF-κB pathway  were found in BRCA1-deficient mammary glands. However, because transcriptional enhancers do not always regulate expression of the most proximal genes, the functional link between BRCA1-affected super-enhancers and their neighboring genes used in our gene ontology analyses need to be experimentally validated. We are also cognizant of the limitation in using the immortalized cell line MCF10A to investigate BRCA1-regulated chromatin events and transcription, which obviously differs from primary breast epithelial cells in vivo. However, the fact that BRCA1 haploinsufficiency displays a similar effect on the selected super-enhancers in clinical samples and MCF10A cells justifies the use of the cell line model for the in-depth mechanistic studies. Moreover, previously published findings using the same MCF10A-based cell culture model have provided physiologically relevant information concerning BRCA1 functions in regulation of epithelial differentiation and maintenance of genome stability [29, 30, 96, 97]. Given the various degrees of functional deficiency of BRCA1 mutations in supporting super-enhancer activity, the in vitro system established in our study could serve as a convenient way of further exploring phenotype-genotype correlation for cancer-predisposing BRCA1 mutations.
How germ-line BRCA1 haploinsufficiency preferentially leads to tissue-specific cancer development remains a longstanding conundrum. Using haploinsufficient HMECs and cell line models, work from several laboratories supports the notion that genomic instability due to compromised BRCA1 activity in replication stress resolution and/or DNA repair contributes to BRCA1-associated tumorigenesis [27, 28, 29, 30, 31, 98]. Of note, Sedic et al. has shown that BRCA1 haploinsufficiency-induced genomic instability occurs specifically in HMECs but not breast fibroblasts , which provides a molecular explanation for tissue-specificity of BRCA1-associated tumorigenesis. However, given the ubiquitous nature of DNA replication stress and DSB DNA repair, it is not clear whether genomic instability alone is sufficient to account for luminal-to-basal transition and subsequent cancer development in BRCA1 mutation carriers. In this regard, mounting evidence suggests that BRCA1-mediated transcriptional regulation plays previously under-appreciated roles in tissue-specific tumor suppression. For example, the alternative NF-κB pathway is constitutively and preferentially active in BRCA1-deficient mammary luminal progenitor cells , the cell of origin for BRCA1-associated tumors. Furthermore, we recently showed that R-loops, transcription byproducts and DNA-RNA hybrids involved in genomic instability, preferentially accumulate in luminal epithelial cells but not in basal or stromal cells of BRCA1 mutation-carrying breast tissue .
Our current study provides a compelling molecular link between BRCA1 haploinsufficiency and deficiency in super-enhancer functions and chromatin looping at a very early stage of BRCA1 mutation-associated breast tumorigenesis. Conceptually, our findings strongly suggest that a direct role of BRCA1 in chromatin reorganization and transcriptional regulation contributes to its tissue-specific tumor suppressor function. A better understanding of the early molecular abnormalities in BRCA1 mutation-carrying breast epithelium could potentially inform development of novel tools to more precisely prevent breast tumors in women with germ-line BRCA1 mutations.
We thank Chi Zhang, Xiayan Zhao, Jerry Chen, Jingwei Li, Fei Ge and Sabrina Smith for technical assistance.
The work was supported by grants to RL from NIH (CA220578); to YH from NIH (CA212674), DOD (W81XWH-17-1-0007); to VXJ from NIH (GM114142, U54CA217297); to CL and RL from NIH (CA214176), and the Cancer Prevention and Research Institute of Texas (CPRIT, RP170126); and to H-CC from an NIH Postdoctoral Training Grant (T32CA148724). ChIP-seq data was generated in the Genome Sequencing Facility, which is supported by NIH-NCI P30 CA054174 (Cancer Center at UT Health San Antonio), NIH Shared Instrument grant 1S10OD021805-01 (S10 grant), and CPRIT Core Facility Award (RP160732).
Availability of data and materials
Sequence data that support the findings of this study have been deposited in NIH Gene Expression Omnibus (GEO) with the accession codes GSE121229. All other remaining data are available within the article, or from the authors upon request.
RL, YH, and VXJ conceived and supervised the project. RL, YH, VXJ, and XZ designed the experiments. XZ, H-CC, and Y-PH performed the experiments. XZ, YW, CL, BHP, VXJ, YH, and RL analyzed the data. XZ and RL wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by Institutional Review Board at the University of Texas Health Science Center at San Antonio with reference number HSC20080058H. All donors signed written consent forms authorizing the use of the specimens.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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