Eda Yildirim

Increased Vascular Smooth Muscle Contractility in TRPC6−/− Mice

ABSTRACT Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6 − / − smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6 −/− smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.

Orai proteins interact with TRPC channels and confer responsiveness to store depletion

The TRPC (C-type transient receptor potential) class of ion channels has been hypothesized to participate in store-operated Ca2+ entry (SOCE). Recently, however, STIM1 and Orai1 proteins have been proposed to form SOCE channels. Whether TRPCs participate in SOCE that is dependent on or regulated by Orai has not been explored. Here we show that Orai1 physically interacts with the N and C termini of TRPC3 and TRPC6, and that in cells overexpressing either TRPC3 or TRPC6 in a store-depletion insensitive manner, these TRPCs become sensitive to store depletion upon expression of an exogenous Orai. Thus, Orai-1, -2, and -3 enhanced thapsigargin-induced calcium entry by 50–150% in cells stably overexpressing either TRPC3 or TRPC6. Orai1 expression had no significant effect on endogenous, thapsigargin-induced calcium entry in wild-type cells (HEK-293, COS1), in HEK cells expressing a thapsigargin-sensitive variant of TRPC3 (TRPC3a), or in HEK cells overexpressing another membrane protein, V1aR. Single-channel cation currents present in membrane patches of TRPC3-overexpressing cells were suppressed by expression of Orai1. We propose that Orai proteins by interacting with TRPCs act as regulatory subunits that confer STIM1-mediated store depletion sensitivity to these channels.

Xist RNA Is a Potent Suppressor of Hematologic Cancer in Mice

X chromosome aneuploidies have long been associated with human cancers, but causality has not been established. In mammals, X chromosome inactivation (XCI) is triggered by Xist RNA to equalize gene expression between the sexes. Here we delete Xist in the blood compartment of mice and demonstrate that mutant females develop a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome (mixed MPN/MDS) with 100% penetrance. Significant disease components include primary myelofibrosis, leukemia, histiocytic sarcoma, and vasculitis. Xist-deficient hematopoietic stem cells (HSCs) show aberrant maturation and age-dependent loss. Reconstitution experiments indicate that MPN/MDS and myelofibrosis are of hematopoietic rather than stromal origin. We propose that Xist loss results in X reactivation and consequent genome-wide changes that lead to cancer, thereby causally linking the X chromosome to cancer in mice. Thus, Xist RNA not only is required to maintain XCI but also suppresses cancer in vivo.

X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription

Dosage compensation in mammals occurs at two levels. In addition to balancing X-chromosome dosage between males and females via X inactivation, mammals also balance dosage of Xs and autosomes. It has been proposed that X-autosome equalization occurs by upregulation of Xa (active X). To investigate mechanism, we perform allele-specific ChIP-seq for chromatin epitopes and analyze RNA-seq data. The hypertranscribed Xa demonstrates enrichment of active chromatin marks relative to autosomes. We derive predictive models for relationships among Pol II occupancy, active mark densities and gene expression, and we suggest that Xa upregulation involves increased transcription initiation and elongation. Enrichment of active marks on Xa does not scale proportionally with transcription output, a disparity explained by nonlinear quantitative dependencies among active histone marks, Pol II occupancy and transcription. Notably, the trend of nonlinear upregulation also occurs on autosomes. Thus, Xa upregulation involves combined increases of active histone marks and Pol II occupancy, without invoking X-specific dependencies between chromatin states and transcription.

The mouse C-type transient receptor potential 2 (TRPC2) channel: Alternative splicing and calmodulin binding to its N terminus

Channels of the C-type transient receptor potential (TRPC) are involved in agonist-stimulated and capacitative calcium entry. There are seven TRPCs, all of which have a Ca2+-dependent calmodulin (CaM)-binding domain in their C termini. We now tested binding of CaM to TRPC N termini and show that only that of TRPC2 binds CaM in a Ca2+-dependent manner. Four TRPC2 cDNAs have been reported: a (also clone 14), b (also clone 17), α, and β. Sequences responsible for CaM binding in TRPC2 a and b are absent from the α and β isoforms. The α and β cDNAs of TRPC2 were reported as alternative forms, when recloning of TRPC2 a and b proved impossible. Here we analyzed total RNA samples from brain and testis for presence of TRPC2 a and b and describe the splicing patterns responsible for their formation, as well as those leading to the α and β forms of TRPC2. We re-assert existence of RNA encoding the TRPC2 a and b, encoded in 21 exons with an initiator ATG in exon 2 for TRPC2a and in exon 4 for TRCP2b. The analysis of α and β TRPC2 cDNAs indicates that although the TRPC2β mRNA may exist, the TRPC2α cDNA is derived from an incompletely processed TRPC2a mRNA: It includes in its presumed 5′-untranslated sequence, 713 nt of TRPC2a cDNA fused to 291 nt of an incompletely excised intron. While encoding an active channel in the mouse, the human TRPC2 appears to be a pseudogene. We searched for the human gene in the data bank and located approximately one-half of it in a chromosomal region syntenic to that of the mouse, with similar intron–exon structure. We conclude that the human TRPC2 gene may never have been an active gene because of incomplete ancestral duplication or, if it was complete at one point, that it became inactive upon loss of chromosomal sequences.

TRPC2: Molecular Biology and Functional Importance

TRPC (canonical transient receptor potential) channels are the closest mammalian homologs of Drosophila TRP and TRP-like channels. TRPCs are rather nonselective Ca2+ permeable cation channels and affect cell functions through their ability to mediate Ca2+ entry into cells and their action to collapse the plasma membrane potentials. In neurons the latter function leads to action potentials. The mammalian genome codes for seven TRPCs of which TRPC2 is the largest with the most restricted pattern of expression and has several alternatively spliced variants. Expressed in model cells, TRPC2 mediates both receptor- and store depletion-triggered Ca2+ entry. TRPC2 is unique among TRPCs in that its complete gene has been lost from the Old World monkey and human genomes, in which its remnants constitute a pseudogene. Physiological roles for TRPC2 have been studied in mature sperm and the vomeronasal sensory system. In sperm, TRPC2 is activated by the sperms interaction with the oocytes zona pellucida, leading to entry of Ca2+ and activation of the acrosome reaction. In the vomeronasal sensory organ (VNO), TRPC2 was found to constitute the transduction channel activated through signaling cascade initiated by the interaction of pheromones with V1R and V2R G protein-coupled receptors on the dendrites of the sensory neurons. V1Rs and V2Rs, the latter working in conjunction with class I MHC molecules, activate G(i)- and G(o)-type G proteins which in turn trigger activation of TRPC2, initiating an axon potential that travels to the axonal terminals. The signal is then projected to the glomeruli of the auxiliary olfactory bulb from where it is carried first to the amygdala and then to higher cortical cognition centers. Immunocytochemistry and gene deletion studies have shown that (1) the V2R-G(o)-MHCIb-beta2m pathway mediates male aggressive behavior in response to pheromones; (2) the V1R-G(i2) pathway mediates mating partner recognition, and (3) these differences have an anatomical correlate in that these functional components are located in anatomically distinct compartments of the VNO. Interestingly, these anatomically segregated signaling pathways use a common transduction channel, TRPC2.

Severely blunted allergen-induced pulmonary Th2 cell response and lung hyperresponsiveness in type 1 transient receptor potential channel-deficient mice

Transient receptor potential channels (TRPCs) are widely expressed and regulate Ca²⁺ entry in the cells that participate in the pathophysiology of airway hyperreactivity, inflammation, and remodeling. In vitro studies point to a role for TRPC1-mediated Ca²⁺ signaling in several of these cell types; however, physiological evidence is lacking. Here we identify TRPC1 signaling as proinflammatory and a regulator of lung hyperresponsiveness during allergen-induced pulmonary response. TRPC1-deficient (Trpc1(-/-)) mice are hyposensitive to methacholine challenge and have significantly reduced allergen-induced pulmonary leukocyte infiltration coupled with an attenuated T helper type 2 (Th2) cell response. Upon in vitro allergen exposure, Trpc1(-/-) splenocytes show impaired proliferation and T cell receptor-induced IL-2 production. A high number of germinal centers in spleens of Trpc1(-/-) mice and elevated levels of immunoglobulins in their serum are indicative of dysregulated B cell function and homeostasis. Thus we propose that TRPC1 signaling is necessary in lymphocyte biology and in regulation of allergen-induced lung hyperresponsiveness, making TRPC1 a potential target for treatment of immune diseases and asthma.

Allelic Imbalance Is a Prevalent and Tissue-Specific Feature of the Mouse Transcriptome

In mammals, several classes of monoallelic genes have been identified, including those subject to X-chromosome inactivation (XCI), genomic imprinting, and random monoallelic expression (RMAE). However, the extent to which these epigenetic phenomena are influenced by underlying genetic variation is unknown. Here we perform a systematic classification of allelic imbalance in mouse hybrids derived from reciprocal crosses of divergent strains. We observe that deviation from balanced biallelic expression is common, occurring in ∼20% of the mouse transcriptome in a given tissue. Allelic imbalance attributed to genotypic variation is by far the most prevalent class and typically is tissue-specific. However, some genotype-based imbalance is maintained across tissues and is associated with greater genetic variation, especially in 5′ and 3′ termini of transcripts. We further identify novel random monoallelic and imprinted genes and find that genotype can modify penetrance of parental origin even in the setting of large imprinted regions. Examination of nascent transcripts in single cells from inbred parental strains reveals that genes showing genotype-based imbalance in hybrids can also exhibit monoallelic expression in isogenic backgrounds. This surprising observation may suggest a competition between alleles and/or reflect the combined impact of cis- and trans-acting variation on expression of a given gene. Our findings provide novel insights into gene regulation and may be relevant to human genetic variation and disease.

Bimodal quantitative relationships between histone modifications for X-linked and autosomal loci

Gene expression is controlled by coordinated action of many epigenetic mechanisms including covalent histone modifications. Although numerous recurrent patterns of colocalized histone modifications have been associated with specific gene expression states, interrelationships between individual modifications are largely unknown. Here, we analyze quantitative relationships between colocalized histone marks during embryonic stem cell (ESC) differentiation and find that, for autosomal genes, these densities follow bimodal patterns. Analysis of repressive H3K27me3 and activating H3K4me3 modifications reveals the expected anticorrelation between them at active promoters but an unexpected positive correlation at inactive promoters. The two trends connect in a region corresponding to bivalent genes. Interestingly, this region is characterized by maximal H3K27 methylation. Resolving gene bivalency during ESC differentiation does not conform to the expected model of two marks as counteracting and competing forces. Although activated genes acquire H3K4me3 and lose H3K27me3, repressed genes lose H3K4me3 without gaining H3K27me3. The behavior of X-linked genes also deviates from expected models. Allele-specific analysis of chromatin modifications during X-chromosome inactivation (XCI) suggests that the silencing machinery focuses on active genes and depletion of H3K4me3 and that H3K27me3 is most significant during establishment of gene silencing. Our analysis reveals nontrivial relationships between H3K4me3 and H3K27me3, reveals unique aspects of gene bivalency, and demonstrates that XCI does not conform neatly to autosomal models.

Genome-wide identification of autosomal genes with allelic imbalance of chromatin state

In mammals, monoallelic gene expression can result from X-chromosome inactivation, genomic imprinting, and random monoallelic expression (RMAE). Epigenetic regulation of RMAE is not fully understood. Here we analyze allelic imbalance in chromatin state of autosomal genes using ChIP-seq in a clonal cell line. We identify approximately 3.7% of autosomal genes that show significant differences between chromatin states of two alleles. Allelic regulation is represented among several functional gene categories including histones, chromatin modifiers, and multiple early developmental regulators. Most cases of allelic skew are produced by quantitative differences between two allelic chromatic states that belong to the same gross type (active, silent, or bivalent). Combinations of allelic states of different types are possible but less frequent. When different chromatin marks are skewed on the same gene, their skew is coordinated as a result of quantitative relationships between these marks on each individual allele. Finally, combination of allele-specific densities of chromatin marks is a quantitative predictor of allelic skew in gene expression.