The epigenetic landscape of mammary gland development and functional differentiation

International audienceMost of the development and functional differentiation in the mammary gland occur after birth. Epigenetics is defined as the stable alterations in gene expression potential that arise during development and proliferation. Epigenetic changes are mediated at the biochemical level by the chromatin conformation initiated by DNA methylation, histone variants, post-translational modifications of histones, non-histone chromatin proteins, and non-coding RNAs. Epigenetics plays a key role in development. However, very little is known about its role in the developing mammary gland or how it might integrate the many signalling pathways involved in mammary gland development and function that have been discovered during the past few decades. An inverse relationship between marks of closed (DNA methylation) or open chromatin (DnaseI hypersensitivity, certain histone modifications) and milk protein gene expression has been documented. Recent studies have shown that during development and functional differentiation, both global and local chromatin changes occur. Locally, chromatin at distal regulatory elements and promoters of milk protein genes gains a more open conformation. Furthermore, changes occur both in looping between regulatory elements and attachment to nuclear matrix. These changes are induced by developmental signals and environmental conditions. Additionally, distinct epigenetic patterns have been identified in mammary gland stem and progenitor cell sub-populations. Together, these findings suggest that epigenetics plays a role in mammary development and function. With the new tools for epigenomics developed in recent years, we now can begin to establish a framework for the role of epigenetics in mammary gland development and disease

Knockdown of <i>mPINC</i> enhances differentiation of HC11 cells.

<p>(A, B) To target <i>mPINC1.0</i> and <i>mPINC1.6</i> (siPINC1.0/1.6), but not <i>DCR2</i>, siRNAs #1 and #2 were used in combination. To target all splice variants (siPINC), siRNAs #1 and #3 were used in combination. (A) Schematic showing siRNA targets of <i>mPINC</i> splice forms. (B) qPCR shows knockdown of <i>mPINC</i> 5 days post-transfection of siRNAs. Target genes were normalized to <i>Gapdh</i> and set relative to levels in the siNEG control transfected HC11 cells. (C, D) Knockdown of <i>mPNC</i> (siPINC1.0/1.6 and siPINC) splice forms increases <i>Wap</i> and <i>Ltf</i>, but not <i>Csn2</i>, expression at 24 (C) and 72 (D) hrs post-hormone induction. Target genes were normalized to <i>Gapdh</i> and set relative to levels in siNEG treated control cells. Data are presented as mean ± SEM (n = 3). (E, F) Knockdown of <i>mPINC</i> also increases dome formation compared to a control. (E) Representative brightfield images show domes following 48 hrs of hormone treatment. Scale bars represent 50 µm. (F) Domes were counted at 48 hours post-hormone treatment from nine 20× fields/experiment and data represent mean ± SEM set relative to the dome number in the siNEG treated control group (n = 6).</p

Microarray identifies potential targets of <i>mPINC</i> in HC11 cells.

<p>(A) Heat map showing transcript profiling of biological replicates of control siNEG cells, siPINC1.0/1.6, and siPINC 5 days post-siRNA transfection ( p<0.01 and fold change >1.8 in either siPINC1.0/1.6 or siPINC relative to siNEG). (B) Graph indicating the most significantly enriched gene ontology terms in the <i>mPINC</i> knockdown data set. (C) Three heat map panels depicting genes that are differentially expressed between either undifferentiated (−LH) <i>mPINC</i> knockdown cells (left panel) or differentiated (+LH) <i>mPINC</i> knockdown cells (middle panel) and differentiated (+LH) <i>mPINC</i> overexpression cells (right panel). 181 genes were found to be differentially regulated by <i>mPINC</i> (141 genes are upregulated by knockdown and downregulated by overexpression, while 40 genes are downregulated by knockdown and upregulated by overexpression at a p<0.01 and fold change >1.4, both for the overexpression and either knockdown group in the opposite direction). Genes whose expression was validated by qPCR are indicated in the heat map on the left. (D) qPCR verified differential expression of genes in <i>mPINC</i> knockdown (KD) compared to overexpression (OE) cells. qPCR was performed on biological triplicates, normalized using <i>Gapdh</i>, and shown as fold change compared to the negative control (either siNEG or LeGO-GFP).</p

<i>mPINC</i> interacts with PRC2 in HC11 cells.

<p>(A–D) RIP assays were performed with HC11 cells using antibodies to PRC2 members including, EZH2, SUZ12 and RbAp46. An MLL1 antibody was used as a negative control and an lncRNA known to interact with PRC2, <i>Tug1</i>, was used as a positive control. RNA was isolated from pull downs to detect associated RNAs. (A) RT-PCR shows <i>mPINC</i> is associated with PRC2 members, but not MLL1. (B–D) qPCR shows the amount of <i>Tug1</i> (B), <i>mPINC1.0</i> (C) and <i>mPINC1.6</i> (D) transcript associated with each protein as a percentage of input RNA levels. Data represent mean ± SD (n = 3).</p

<i>mPINC</i> expression peaks in the late pregnant and early involuting gland.

<p>(A) RT-PCR shows multiple splice forms of <i>mPINC1.0</i> and <i>mPINC1.6</i> are expressed during mammary gland development. Primers designed to the extreme ends of <i>mPINC1.0</i> and <i>mPINC1.6</i> were used to amplify cDNA from mammary gland developmental stages. (w Vir.: weeks old virgin, d Preg.: days pregnancy, d Lac.: days lactation, d Inv.: days involution). PCR products were sequenced and found to be new splice forms of <i>mPINC</i>, including a new splice variant of <i>mPINC1.6</i> called <i>DCR2</i>, for deleted conserved region 2. (B) Schematic diagram of <i>mPINC</i> exonic structure. Black boxes represent exons that are always included, grey boxes are sometimes included and clear boxes are never included. Nucleotide length is indicated above each exon along with black lines that overlap the most conserved regions of the <i>PINC</i> locus, CR1 and CR2. Exon 6 sometimes has an additional 24 nucleotides at the 3′ end in the <i>mPINC1.0</i> and <i>mPINC1.6</i> splice forms. This alternative splice site does not correlate with the inclusion/exclusion of any particular exon and its function is unknown. (C) qPCR shows <i>mPINC</i> is highest during late pregnancy and early involution. Mammary glands were harvested from 3 female Balb/c mice for each stage (V: adult virgin, dP: days pregnant, dL: days lactation, dI: days involution). Target genes were normalized to <i>Actb</i> and set relative to levels in the virgin mammary gland. (D) <i>mPINC</i> expression is most abundant in the mammary gland. Tissues were harvested from three 10 week old virgin Balb/c female mice and testis, epididymis, and prostate was harvested from three 12 week old male Balb/c mice. ND indicates tissues in which <i>mPINC</i> was not detected by qPCR. Target genes were normalized to <i>Actb</i> and set relative to levels in the lung.</p

<i>mPINC</i> rises specifically in the luminal compartment during pregnancy and is enriched in luminal and alveolar progenitors of the mammary gland.

<p>(A–C) RNA was isolated from FACs sorted mammary populations including, virgin luminal (VL) and basal (VB) as well as pregnant luminal (PL) and basal (PB). (A) qPCR showed <i>Krt8</i> expression was enriched in the luminal populations (VL and PL), (B) <i>Krt14</i> was enriched in the basal populations (VB and PB) and (C) <i>mPINC</i> was enriched in the pregnant luminal population, (D–F) MECs were FACs sorted into luminal and basal populations using CD24 and CD29. The luminal population was selected and further sorted into mature luminal (ML), luminal progenitors (LP), and alveolar progenitors (AP) using CD14 and ckit. (D) FACs dot plots showing CD24 and CD29 (left panel) as well as CD14 and ckit (right panel) from virgin MECs. (E) qPCR showed <i>Ly6a</i> and <i>Wnt4</i> enriched in the ML population and <i>Elf5</i> in the LP population thus verifying the purity of each population. (F) <i>mPINC</i> was enriched in the luminal progenitors and alveolar progenitors. Data represent mean ±SD (n = 3). Target genes were normalized to <i>Gapdh</i>.</p

Overexpressed <i>mPINC</i> transcripts interact with PRC2 in HC11 cells.

<p>(A–D) RIP assays were performed with <i>mPINC</i> overexpressing HC11 cell using antibodies to PRC2 members including, EZH2, SUZ12 and RbAp46. An MLL1 antibody was used as a negative control. (A) RT-PCR shows that overexpressed <i>mPINC1.0</i>, <i>mPINC1.6</i> and the <i>DCR</i> mutant interact with PRC2 members, but not IgG or MLL1. (B–D) qPCR shows the amount of <i>mPINC1.0</i> (B), <i>mPINC1.6</i> (C) and DCR (D) transcript associated with each protein as a percentage of input RNA levels. Data represent mean ± SD (n = 3).</p

<i>mPINC</i> is enriched in alveolar cells of the pregnant gland.

<p><i>In situ</i> hybridization using DIG-labeled probes shows <i>mPINC1.0</i> and <i>mPINC1.6</i> are expressed in alveolar cells of the 12 day mouse mammary gland. Sense control probes for <i>mPINC1.0</i> and <i>mPINC1.6</i> show very little signal in the bottom panels. Scale bars represent 100 µm.</p

<i>mPINC</i> associates with PRC2 in the 16-day pregnant mammary gland.

<p>(A–D) RIP assays were performed with MECs purified from mammary glands at day 16 of pregnancy using antibodies to PRC2 members and its associated histone modification, H3meK27. Antibodies to MLL1 and its associated histone modification, H3meK4, were also used as negative controls. (A) RT-PCR shows <i>mPINC</i> is associated with PRC2 members and H3meK27. <i>mPINC</i> does not associate with MLL1 or H3meK4. (B–D) qPCR shows fold enrichment of <i>mPINC</i> transcript levels associated with EZH2 (B), SUZ12 (C) RpAp46 (D) and MLL1 (E) relative to <i>Gapdh</i> levels. Data represent mean ± SD (n = 3).</p