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      Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

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          Abstract

          HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences ( CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

          Author Summary

          NOTCH signaling is a central developmental pathway that influences a multitude of cell fate decisions and differentiation steps as well as later tissue homeostasis and regeneration. The three HEY genes encode basic helix-loop-helix transcription factors that are critical effectors to convey signaling by NOTCH receptors and similar signaling systems. This is underscored by the multitude of developmental defects observed in HEY single- and double-mutant mice. The mode of action of HEY proteins remained largely unexplored, however. By gene expression analysis and chromatin immunoprecipitation we have now identified a large set of HEY target genes. While only 500–2,000 mRNAs are regulated by HEY1 or HEY2, there are around 10,000 binding sites in the genome. HEY proteins act as transcriptional repressors that bind close to transcriptional start sites in all cases tested. In contrast, gene activation seems to be mediated by indirect/secondary mechanisms. The extent of regulation is rather limited, implicating HEY genes in modulating rather than switching on or off target gene expression. All our in vitro and in vivo data point to a high degree of redundancy between the three HEY genes, suggesting that tissue specific patterns and expression levels determine the final outcome of HEY induced cellular responses.

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          A chromatin landmark and transcription initiation at most promoters in human cells.

          We describe the results of a genome-wide analysis of human cells that suggests that most protein-coding genes, including most genes thought to be transcriptionally inactive, experience transcription initiation. We found that nucleosomes with H3K4me3 and H3K9,14Ac modifications, together with RNA polymerase II, occupy the promoters of most protein-coding genes in human embryonic stem cells. Only a subset of these genes produce detectable full-length transcripts and are occupied by nucleosomes with H3K36me3 modifications, a hallmark of elongation. The other genes experience transcription initiation but show no evidence of elongation, suggesting that they are predominantly regulated at postinitiation steps. Genes encoding most developmental regulators fall into this group. Our results also identify a class of genes that are excluded from experiencing transcription initiation, at which mechanisms that prevent initiation must predominate. These observations extend to differentiated cells, suggesting that transcription initiation at most genes is a general phenomenon in human cells.
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            Variance stabilization applied to microarray data calibration and to the quantification of differential expression.

            We introduce a statistical model for microarray gene expression data that comprises data calibration, the quantification of differential expression, and the quantification of measurement error. In particular, we derive a transformation h for intensity measurements, and a difference statistic Deltah whose variance is approximately constant along the whole intensity range. This forms a basis for statistical inference from microarray data, and provides a rational data pre-processing strategy for multivariate analyses. For the transformation h, the parametric form h(x)=arsinh(a+bx) is derived from a model of the variance-versus-mean dependence for microarray intensity data, using the method of variance stabilizing transformations. For large intensities, h coincides with the logarithmic transformation, and Deltah with the log-ratio. The parameters of h together with those of the calibration between experiments are estimated with a robust variant of maximum-likelihood estimation. We demonstrate our approach on data sets from different experimental platforms, including two-colour cDNA arrays and a series of Affymetrix oligonucleotide arrays.
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              HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.

              We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                May 2012
                May 2012
                17 May 2012
                : 8
                : 5
                : e1002728
                Affiliations
                [1 ]Developmental Biochemistry, Theodor-Boveri-Institute, Biocenter, University of Wuerzburg, Wuerzburg, Germany
                [2 ]Laboratory for Microarray Applications, and Physiological Chemistry I, Theodor-Boveri-Institute, Biocenter, University of Wuerzburg, Wuerzburg, Germany
                [3 ]Genome Institute of Singapore, Singapore, Singapore
                [4 ]Biochemistry and Molecular Biology, Theodor-Boveri-Institute, Biocenter, University of Wuerzburg, Wuerzburg, Germany
                University of Cambridge, United Kingdom
                Author notes

                ¤: Current address: Joint Genome Institute, Walnut Creek, California, United States of America

                Conceived and designed the experiments: JH DW C-LW MG. Performed the experiments: JH DW EE AW SK W-KS EW. Analyzed the data: JH DW EE C-LW ME MG. Wrote the paper: JH DW MG.

                Article
                PGENETICS-D-11-02394
                10.1371/journal.pgen.1002728
                3355086
                22615585
                012e8754-257c-4c50-bd33-01bfbcc05cff
                Heisig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 5 November 2011
                : 5 April 2012
                Page count
                Pages: 14
                Categories
                Research Article
                Biology
                Computational Biology
                Genomics
                Functional Genomics
                Genome Expression Analysis
                Molecular Genetics
                Gene Regulation
                Gene Expression
                Microarrays
                Sequence Analysis
                Developmental Biology
                Molecular Development
                Signaling
                Organism Development
                Organogenesis
                Cell Fate Determination
                Genetics
                Epigenetics
                Chromatin
                Gene Expression
                DNA transcription
                Chromatin
                Molecular Genetics
                Gene Regulation
                Gene Function
                Genomics
                Functional Genomics
                Genome Expression Analysis
                Genome Sequencing
                Molecular Cell Biology
                Gene Expression
                Nucleic Acids
                Signal Transduction
                Medicine
                Cardiovascular
                Congenital Heart Disease
                Vascular Biology

                Genetics
                Genetics

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