Nuclei contain two differentially regulated pools of diacylglycerol

AbstractA number of recent studies have highlighted the presence of a nuclear pool of inositol lipids [1,2] that is regulated during progression through the cell cycle [1,3], differentiation [1,2] and after DNA damage [2], suggesting that a number of different regulatory pathways impinge upon this pool of lipids. It has been suggested that the downstream consequence of the activation of one of these nuclear phosphoinositide (PI) regulatory pathways is the generation of nuclear diacylglycerol (DAG) [1,3,4], which is important in the activation of nuclear protein kinase C (PKC) [5–7]. Activation of PKC in turn appears to regulate the progression of cells through G1 and into S phase [4] and through G2 to mitosis [3,8–11]. Although the evidence is enticing, there is as yet no direct demonstration that nuclear PIs can be hydrolysed to generate nuclear DAG. Previous data in murine erythroleukemia (MEL) cells have suggested that nuclear phosphoinositidase Cβ1 (PIC-β1) activity is important in the generation of nuclear DAG. Here, we demonstrate that the molecular species of nuclear DAG bears little resemblance to the PI pool and is unlikely to be generated directly by hydrolysis of these inositol lipids. Further, we show that there are in fact two distinct subnuclear pools of DAG; one that is highly disaturated and mono-unsaturated (representing more than 90% of the total nuclear DAG) and one that is highly polyunsaturated and is likely to be derived from the hydrolysis of PI. Analysis of these pools, either after differentiation or during cell-cycle progression, suggests that the pools are independently regulated, possibly by the regulation of two different nuclear phospholipase Cs (PLCs)

Epigenetic homogeneity in histone methylation underlies sperm programming for embryonic transcription

Abstract: Sperm contributes genetic and epigenetic information to the embryo to efficiently support development. However, the mechanism underlying such developmental competence remains elusive. Here, we investigated whether all sperm cells have a common epigenetic configuration that primes transcriptional program for embryonic development. Using calibrated ChIP-seq, we show that remodelling of histones during spermiogenesis results in the retention of methylated histone H3 at the same genomic location in most sperm cell. This homogeneously methylated fraction of histone H3 in the sperm genome is maintained during early embryonic replication. Such methylated histone fraction resisting post-fertilisation reprogramming marks developmental genes whose expression is perturbed upon experimental reduction of histone methylation. A similar homogeneously methylated histone H3 fraction is detected in human sperm. Altogether, we uncover a conserved mechanism of paternal epigenetic information transmission to the embryo through the homogeneous retention of methylated histone in a sperm cells population

Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance

Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance

RIME proteomics of estrogen and progesterone receptors in breast cancer

Nuclear receptors play an important role in transcriptional regulation of diverse cellular processes and is also relevant in diseases such as cancer. In breast cancer, the nuclear receptors – estrogen receptor (ER) and progesterone receptor (PR) are classical markers of the disease and are used to classify breast cancer subtypes. Using a recently developed affinity purification MS technique (RIME) [1], we investigate the protein interactors of ER and PR in breast cancer cell lines upon stimulation by the ligands – estrogen and progesterone. The data is deposited at proteomeXchange (PXD002104) and is part of a publication [2] that explains the link between the two nuclear receptors and potential consequences of this in breast cancer. In this manuscript, we describe the methodology used and provide details on experimental procedures, analysis methods and analysis of raw data. The purpose of this article is to enable reproducibility of the data and provide technical recommendations on performing RIME in hormonal contexts

Copy number aberrations drive kinase rewiring, leading to genetic vulnerabilities in cancer

Somatic DNA copy number variations (CNVs) are prevalent in cancer and can drive cancer progression, albeit with often uncharacterized roles in altering cell signaling states. Here, we integrate genomic and proteomic data for 5,598 tumor samples to identify CNVs leading to aberrant signal transduction. The resulting associations recapitulate known kinase-substrate relationships, and further network analysis prioritizes likely causal genes. Of the 303 significant associations we identify from the pan-tumor analysis, 43% are replicated in cancer cell lines, including 44 robust gene-phosphosite associations identified across multiple tumor types. Several predicted regulators of hippo signaling are experimentally validated. Using RNAi, CRISPR, and drug screening data, we find evidence of kinase addiction in cancer cell lines, identifying inhibitors for targeting of kinase-dependent cell lines. We propose copy number status of genes as a useful predictor of differential impact of kinase inhibition, a strategy that may be of use in the future for anticancer therapies.ISSN:2666-3864ISSN:2211-124

Interaction of the Type Iα PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4,5-bisphosphate in the regulation of PLD2 activity

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P(2): phosphatidylinositol 4-phosphate 5-kinase α (Type Iα PIPkinase). We find that both PLD1 and PLD2 interact with the Type Iα PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Iα PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P(2) is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid