Histone modification profiling in breast cancer cell lines highlights commonalities and differences among subtypes

Abstract Background Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy. Results To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells. Discussion Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway. Conclusions Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment.https://deepblue.lib.umich.edu/bitstream/2027.42/142394/1/12864_2018_Article_4533.pd

Histone modification profiling in breast cancer cell lines highlights commonalities and differences among subtypes

Abstract Background Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy. Results To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells. Discussion Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway. Conclusions Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment

p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G1 phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state

p53 regulates cell cycle and microRNAs to promote differentiation of human embryonic stem cells.

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state

Genome-wide CRISPR-Cas9 screen analyzed by SLIDER identifies network of repressor complexes that regulate TRIM24

Summary: TRIM24 is an oncogenic chromatin reader that is frequently overexpressed in human tumors and associated with poor prognosis. However, TRIM24 is rarely mutated, duplicated, or rearranged in cancer. This raises questions about how TRIM24 is regulated and what changes in its regulation are responsible for its overexpression. Here, we perform a genome-wide CRISPR-Cas9 screen by fluorescence-activated cell sorting (FACS) that nominated 220 negative regulators and elucidated a regulatory network that includes the KAP1 corepressor, CNOT deadenylase, and GID/CTLH E3 ligase. Knocking out required components of these three complexes caused TRIM24 overexpression, confirming their negative regulation of TRIM24. Our findings identify regulators of TRIM24 that nominate previously unexplored contexts for this oncoprotein in biology and disease. These findings were enabled by SLIDER, a new scoring system designed and vetted in our study as a broadly applicable tool for analysis of CRISPR screens performed by FACS

p53 protein is induced during differentiation of hESCs.

<p>(A) qRT-PCR. RNA from hESCs treated with RA in medium without FGF for 5 d (R0–R5), were subjected to qRT-PCR assay (data are presented as mean ± SEM). (B and C) Western blot. Lysates (total cell lysate [TCL]) prepared from hESCs cultured as in (A) were analyzed by Western blotting, the blots in (B) were quantitated (C): the average density of three different blots is plotted (*, <i>p</i><0.05). (D) <i>TP53</i> qRT-PCR. RNA samples prepared as in (A) were subjected to qRT-PCR assay (mean ± SEM). (E) Immunofluorescence. hESCs in complete CM or treated with RA for 3 d were stained with antibodies against p53 and OCT4, and nuclei were stained with DAPI. Scale bar is 50 µm. (F) p53 nuclear localization. Nuclear extracts prepared from hESCs cultured as in (A) were analyzed by Western blotting. (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s001" target="_blank">Figure S1</a>.).</p

Acetylation of Lys373 leads to stabilization of p53.

<p>(A) p53 acetylation. Equal amounts of p53 were immunoprecipitated by adjusting the amounts of total cell lysates prepared from hESCs and probed with p53K373ac antibody. (B) Immunofluorescence. hESCs treated with RA for 3 d were stained with antibodies against p53K373ac and OCT4; nuclei were stained with DAPI. (C) Co-immunoprecipitation. Cell lysates from RA-treated hESCs were immunoprecipitated with p53 followed by Western blotting. (D) p53 acetylation. p53 immunoprecipitated from hESCs cultured under differentiating conditions and treated with either circumin on day 2 or nicotinamide on day 4 and probed with p53K373ac antibody. (E) Co-immunoprecipitation. Cell lysates from differentiating hESCs were immunoprecipitated with HDM2 or TRIM24 antibodies and analyzed by Western blotting. (F) Endogenous p53 ubiquitination. hESCs cultured under differentiating conditions were treated with MG132 + RA or MG132 + Adr; endogenous p53 was immunoprecipitated and probed for ubiquitin (top panel). Same blot was reprobed with p53 antibody to show the equal p53 pull down (bottom panel). (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s002" target="_blank">Figure S2</a>.) IP, mmunoprecipitation; Ub, ubiquitin; WB, Western blot.</p

p53 drives differentiation of hESCs.

<p>(A) AP staining. hESCs transfected with non-target (siControl) or siRNA specific to p53 (<i>siTP53</i>) or p21 (<i>siCDKN1A</i>) were treated with RA and stained for AP (blue colonies). (B and C) hESCs transfected and treated as in (A) were used in Western blotting (B) and qRT-PCR (C) analyses. The blots in (B) were quantitated, and average density of three different blots is plotted (bottom panel) (*, <i>p</i><0.05) (mean ± SEM). (D) OCT4 + SSEA4 staining. hESCs transfected with siRNA followed by RA treatment were stained for SSEA4 and OCT4 and subjected to dual flow cytometry analysis. Triplicate samples were analyzed in each experiment, and data analyzed with FACSDiva software. Decreases in fraction of OCT4-positive cells, as compared to siControl untreated, are indicated in red. (E) AP staining. hESCs transfected with <i>HDM2</i> or <i>TRIM24</i> siRNA were treated with RA and stained for AP. (F) Quantified AP-stained colonies. Date shown are for 50 colonies per treatment in three separate experiments (in [A] and [E]), scored as undifferentiated, partially differentiated, or fully differentiated colonies, mean ± SEM. (G) OCT4 + SSEA4 staining and flow cytometry analysis as in (D) after transfection with siRNA targeting <i>HDM2</i> or <i>TRIM24</i>. (H) Cell cycle analysis. hESCs transfected with <i>HDM2</i> or <i>TRIM24</i> siRNA were stained with PI and subjected to cell cycle analysis. (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s003" target="_blank">Figures S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s005" target="_blank">S5</a>.).</p

The consequence of p53 accumulation in hESCs.

<p>(A) Cell cycle analysis. hESCs transfected with non-target (siControl) or siRNA specific to p53 (<i>siTP53</i>) or p21 (<i>siCDKN1A</i>) and treated with RA were stained with PI and subjected to flow cytometry analysis to determine DNA content. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#s2" target="_blank">Results</a> quantitated as fold change in cell cycle are shown. (B) qRT-PCR. RNA from hESCs treated with RA for 4 d or Adr for 6 h were subjected to qRT-PCR assay using primers specific for human <i>CDKN1A</i>. (C) ChIP. p53-bound chromatin was immunoprecipitated from hESCs, and p53 enrichment on <i>CDKN1A</i> was analyzed by qRT-PCR using primers encompassing p53REs (*, <i>p</i><0.05). Scheme representing location of p53RE and primers used for ChIP-qRT-PCR are shown on the top (asterisk indicates the 3′ end of the gene). (D) hESCs treated with RA or Adr were lysed, and cell lysates were blotted for γ-H2AX. (E) Apoptosis assay. hESCs treated as in (D) were stained with Annexin V and PI, and percent apoptotic cells was determined by flow cytometry (mean ± SEM). (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s003" target="_blank">Figures S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s004" target="_blank">S4</a>.).</p