Growth hormone-STAT5 regulation of growth, hepatocellular carcinoma, and liver metabolism.

Baik, Myunggi,Yu, Ji Hoon,Hennighausen, Lothar New York Academy of Sciences 2011 Annals of the New York Academy of Sciences Vol.1229 No.1

<P>The liver is a primary target of growth hormone (GH). GH signals are mediated by the transcription factor signal transducer and activator of transcription 5 (STAT5). Here, we focus on recent discoveries about the role of GH-STAT5 signaling in hepatic physiology and pathophysiology. We discuss roles of the GH-STAT5 axis in body growth, lipid metabolism, and the cell cycle pertaining to hepatosteatosis, fibrosis, and hepatocellular carcinoma. Finally, we discuss recent discoveries about the role of GH-STAT5 in sex-specific gene expression and bile acid, steroid, and drug metabolism.</P>

Octopus-toolkit: a workflow to automate mining of public epigenomic and transcriptomic next-generation sequencing data

Kim, Taemook,Seo, Hogyu David,Hennighausen, Lothar,Lee, Daeyoup,Kang, Keunsoo Oxford University Press 2018 Nucleic acids research Vol.46 No.9

<P><B>Abstract</B></P><P>Octopus-toolkit is a stand-alone application for retrieving and processing large sets of next-generation sequencing (NGS) data with a single step. Octopus-toolkit is an automated set-up-and-analysis pipeline utilizing the Aspera, SRA Toolkit, FastQC, Trimmomatic, HISAT2, STAR, Samtools, and HOMER applications. All the applications are installed on the user's computer when the program starts. Upon the installation, it can automatically retrieve original files of various epigenomic and transcriptomic data sets, including ChIP-seq, ATAC-seq, DNase-seq, MeDIP-seq, MNase-seq and RNA-seq, from the gene expression omnibus data repository. The downloaded files can then be sequentially processed to generate BAM and BigWig files, which are used for advanced analyses and visualization. Currently, it can process NGS data from popular model genomes such as, human (<I>Homo sapiens</I>), mouse (<I>Mus musculus</I>), dog (<I>Canis lupus familiaris</I>), plant (<I>Arabidopsis thaliana</I>), zebrafish (<I>Danio rerio</I>), fruit fly (<I>Drosophila melanogaster</I>), worm (<I>Caenorhabditis elegans</I>), and budding yeast (<I>Saccharomyces cerevisiae</I>) genomes. With the processed files from Octopus-toolkit, the meta-analysis of various data sets, motif searches for DNA-binding proteins, and the identification of differentially expressed genes and/or protein-binding sites can be easily conducted with few commands by users. Overall, Octopus-toolkit facilitates the systematic and integrative analysis of available epigenomic and transcriptomic NGS big data.</P>

Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

Yamaji, Daisuke,Kang, Keunsoo,Robinson, Gertraud W.,Hennighausen, Lothar Oxford University Press 2013 Nucleic acids research Vol.41 No.3

<P>The transcription factors Signal Transducer and Activator of Transcription (STAT) 5A/B mediate prolactin-induced mammary development during pregnancy. However, it is not clear how the different processes, expansion and maturation of alveolar precursor cells and the differential induction of milk protein genes are regulated on a molecular level. We have used mouse genetics and genome-wide analyses to determine how altering concentrations of STAT5A and STAT5B impacts mammary epithelial development during pregnancy and the regulation of target genes. The presence of only a single <I>Stat5a</I> or <I>Stat5b</I> allele was sufficient for the establishment of histologically undifferentiated alveolar units and two alleles permitted the execution of a differentiation program similar to that found with all four alleles. While one copy of <I>Stat5</I> induced limited expression of target genes, two copies activated a lactation-like gene signature. Using ChIP-seq analyses on intact tissue under physiological conditions, we found that highly expressed and regulated genes were bound by STAT5 in their promoter proximal regions, whereas upstream binding had minor biological consequences. Remarkably, 80% of the genes bound by STAT5 <I>in vivo </I>were not under STAT5 control. RNA polymerase II intensity was directly proportional to STAT5 concentration only on STAT5 regulated genes providing mechanistic insight by which STAT5 activates mammary specific genes.</P>

The interdependence of mammary-specific superenhancers and their native promoters facilitates gene activation during pregnancy

Xianke Zeng,Hye Kyung Lee,Chaochen Wang,Precious Achikeh,Chengyu Liu,Lothar Hennighausen 생화학분자생물학회 2020 Experimental and molecular medicine Vol.52 No.-

Lineage-specific genetic programs rely on cell-restricted super-enhancers, which are platforms for high-density transcription factor occupation. It is not known whether super-enhancers synergize specifically with their native promoters or provide autonomous and independent regulatory platforms. Here, we investigated the ability of the mammary Wap super-enhancer to activate the promoter of the juxtaposed and ubiquitously expressed Tbrg4 gene in the mouse mammary gland. The Wap super-enhancer was fused, alone or in combination with the Wap promoter, to the Tbrg4 gene. While the super-enhancer increased the expression of the Tbrg4 promoter five-fold, the combination of the super-enhancer and promoter resulted in 80-fold gene upregulation, demonstrating lineage-specific promoter–enhancer synergy. Employing ChIP-seq profiling to determine transcription factor binding and identify activating histone marks, we uncovered a chromatin platform that enables the high-level expression of the native promoter–enhancer but not the heterologous promoter. Taken together, our data reveal that lineage-specific enhancer–promoter synergy is critical for mammary gene regulation during pregnancy and lactation.

Poster Session : PS 1454 ; Hemato-Oncology(Oncology) : The Methyltransferases EZH1 and EZH2 Control Hepatocyte Homeostasis and Regeneration

( Minjee Kim ),( Woo Kyun Bae ),( Valentina M Factor ),( Keunsoo Kang ),( Lothar Hennighausen ) 대한내과학회 2014 대한내과학회 추계학술발표논문집 Vol.2014 No.1

Background: Polycomb group protein enhancer of zeste homolog 2 (EZH2) controls cell proliferation and differentiation and is frequently up-regulated in hepatocellular carcinoma. Genetic inactivation of Ezh2 did not produce obvious phenotypic differences in knockout livers suggesting a functional redundancy with Ezh1 homolog. Methods: To investigate the role of both Ezh1 and Ezh2 in liver homeostasis, we generated double knockout mice (E1/2KO) by crossing Ezh1 null and albumin promoter- driven Ezh2 conditional-null mice.The combined loss of EZH1 and EZH2 in mouse hepatocytes caused a global and gene-specific depletion of H3K27me3 marks including genes associated with hepatocyte homeostasis and regeneration. Mice mutant for both Ezh1 and Ezh2 remained viable and overtly normal by 3 months of age. Thereafter, E1/2KO mice exhibited progressive liver abnormalities manifested by development of regenerative nodules and concomitant periportal fibrosis, infiammatory infiltration and activation of A6-positive hepatic progenitor cells (HPCs). Results: In response to chronic treatment with CCl4, E1/2KO mice showed increased hepatic degeneration associated with liver dysfunction, cholestasis, and reduced ability to proliferate. CCl4-induced hepatotoxicity triggered an activation of HPCs, which did not prevent early lethality of E1/2KO mice. After 2/3ds partial hepatectomy, E1/2KO livers showed an increased liver injury and a blunted regenerative response. RNA-seq analysis revealed dysregulation of genes related to regulation of cell survival, fibrosis, and proliferation including Cdkn2a and Cdkn2b. Conclusions: This work demonstrates a critical role of EZH1 and EZH2in maintaining hepatic homeostasis and ability to regenerate.

Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice

Lee, Hye Kyung,Willi, Michaela,Wang, Chaochen,Yang, Chul Min,Smith, Harold E.,Liu, Chengyu,Hennighausen, Lothar Oxford University Press 2017 Nucleic acids research Vol.45 No.8

<P><B>Abstract</B></P><P>The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the <I>Csn1s1</I> mammary enhancer from neighboring genes resulted in the activation of <I>Sult1d1</I> at a distance of more than 95 kb but not the more proximal and silent <I>Sult1e1</I> gene. Loss of this CTCF site led to <I>de novo</I> interactions between the <I>Sult1d1</I> promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable <I>in vivo</I> activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.</P>

The transcription factors signal transducer and activator of transcription 5A (STAT5A) and STAT5B negatively regulate cell proliferation through the activation of cyclin‐dependent kinase inhibitor 2b (<i>Cdkn2b</i>) and <i>Cdkn1a</i> expression

Yu, Ji Hoon,Zhu, Bing‐,Mei,Wickre, Mark,Riedlinger, Gregory,Chen, Weiping,Hosui, Atsushi,Robinson, Gertraud W.,Hennighausen, Lothar Wiley Subscription Services, Inc., A Wiley Company 2010 Hepatology Vol.52 No.5

<P><B>Abstract</B></P><P>Although the cytokine‐inducible transcription factor signal transducer and activator of transcription 5 (STAT5) promotes proliferation of a wide range of cell types, there are cell‐specific and context‐specific cases in which loss of STAT5 results in enhanced cell proliferation. Here, we report that loss of STAT5 from mouse embryonic fibroblasts (MEFs) leads to enhanced proliferation, which was linked to reduced levels of the cell cycle inhibitors p15<SUP>INK4B</SUP> and p21<SUP>CIP1</SUP>. We further demonstrate that growth hormone, through the transcription factor STAT5, enhances expression of the <I>Cdkn2b</I> (cyclin‐dependent kinase inhibitor 2B) gene and that STAT5A binds to interferon‐gamma–activated sequence sites within the promoter. We recently demonstrated that ablation of STAT5 from liver results in hepatocellular carcinoma upon CCl<SUB>4</SUB> treatment. We now establish that STAT5, like in MEFs, activates expression of the <I>Cdkn2b</I> gene in liver tissue. Loss of STAT5 led to diminished p15<SUP>INK4B</SUP> and increased hepatocyte proliferation. <I>Conclusion:</I> This study for the first time demonstrates that cytokines, through STAT5, induce the expression of a key cell cycle inhibitor. These experiments therefore shed mechanistic light on the context‐specific role of STAT5 as tumor suppressor. (HEPATOLOGY 2010;52:1808‐1818)</P>

The gene encoding the hematopoietic stem cell regulator CCN3/NOV is under direct cytokine control through the transcription factors STAT5A/B.

Kimura, Akiko,Martin, Cyril,Robinson, Gertraud W,Simone, James M,Chen, Weiping,Wickre, Mark C,O'Shea, John J,Hennighausen, Lothar American Society for Biochemistry and Molecular Bi 2010 The Journal of biological chemistry Vol.285 No.43

<P>Cytokines control the biology of hematopoietic stem cells (HSCs) and progenitor cells in part through the transcription factors STAT5A/B. To investigate the target genes of STAT5A/B activated by cytokines in HSCs and progenitors, we performed microarray analyses using Lineage(-) Sca-1(+) c-Kit(+) (KSL) cells in the presence and absence of STAT5A/B. Stimulation with a mixture containing IL-3, IL-6, stem cell factor, thrombopoietin, and Flt3 ligand induced Ccn3/Nov mRNA over 100-fold in WT (control) but not Stat5a/b-null KSL cells. CCN3/NOV is a positive regulator of human HSC self-renewal and development of committed blood cells. Without stimulation, the Ccn3/Nov signal level was low in control KSL cells similar to Stat5a/b-null KSL cells. To determine which cytokine activates the Ccn3/Nov gene, we analyzed Lineage(-) c-Kit(+) (KL) and 32D cells using quantitative PCR and ChIP assays. Although stimulation with a mixture lacking IL-3 prevented the induction of Ccn3/Nov in control KL cells, IL-3 alone could induce Ccn3/Nov mRNA in control KL and 32D cells. ChIP assays using 32D cells revealed IL-3-induced binding of STAT5A/B to a γ-interferon-activated sequences site in the Ccn3/Nov gene promoter. This is the first report that Ccn3/Nov is directly induced by cytokines through STAT5A/B.</P>

Metformin Inhibits Growth Hormone–Mediated Hepatic <i>PDK4</i> Gene Expression Through Induction of Orphan Nuclear Receptor Small Heterodimer Partner

Kim, Yong Deuk,Kim, Yong-Hoon,Tadi, Surendar,Yu, Ji Hoon,Yim, Yong-Hyeon,Jeoung, Nam Ho,Shong, Minho,Hennighausen, Lothar,Harris, Robert A.,Lee, In-Kyu,Lee, Chul-Ho,Choi, Hueng-Sik American Diabetes Association 2012 Diabetes Vol.61 No.10

<P><B/></P><P>Growth hormone (GH) is a counter-regulatory hormone that plays an important role in preventing hypoglycemia during fasting. Because inhibition of the pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinase 4 (PDK4) conserves substrates for gluconeogenesis, we tested whether GH increases PDK4 expression in liver by a signaling pathway sensitive to inhibition by metformin. The effects of GH and metformin were determined in the liver of wild-type, small heterodimer partner (SHP)-, PDK4-, and signal transducer and activator of transcription 5 (STAT5)-null mice. Administration of GH in vivo increased PDK4 expression via a pathway dependent on STAT5 phosphorylation. Metformin inhibited the induction of PDK4 expression by GH via a pathway dependent on AMP-activated protein kinase (AMPK) and SHP induction. The increase in PDK4 expression and PDC phosphorylation by GH was reduced in STAT5-null mice. Metformin decreased GH-mediated induction of PDK4 expression and metabolites in wild-type but not in SHP-null mice. In primary hepatocytes, dominant-negative mutant-AMPK and SHP knockdown prevented the inhibitory effect of metformin on GH-stimulated PDK4 expression. SHP directly inhibited STAT5 association on the <I>PDK4</I> gene promoter. Metformin inhibits GH-induced PDK4 expression and metabolites via an AMPK-SHP–dependent pathway. The metformin-AMPK-SHP network may provide a novel therapeutic approach for the treatment of hepatic metabolic disorders induced by the GH-mediated pathway.</P>

Genome-wide analyses reveal the extent of opportunistic STAT5 binding that does not yield transcriptional activation of neighboring genes

Zhu, Bing-Mei,Kang, Keunsoo,Yu, Ji Hoon,Chen, Weiping,Smith, Harold E.,Lee, Daeyoup,Sun, Hong-Wei,Wei, Lai,Hennighausen, Lothar Oxford University Press 2012 Nucleic acids research Vol.40 No.10

<P>Signal Transducers and Activators of Transcription (STAT) 5A/B regulate cytokine-inducible genes upon binding to GAS motifs. It is not known what percentage of genes with GAS motifs bind to and are regulated by STAT5. Moreover, it is not clear whether genome-wide STAT5 binding is modulated by its concentration. To clarify these issues we established genome-wide STAT5 binding upon growth hormone (GH) stimulation of wild-type (WT) mouse embryonic fibroblasts (MEFs) and MEFs overexpressing STAT5A more than 20-fold. Upon GH stimulation, 23 827 and 111 939 STAT5A binding sites were detected in WT and STAT5A overexpressing MEFs, respectively. 13 278 and 71 561 peaks contained at least one GAS motif. 1586 and 8613 binding sites were located within 2.5 kb of promoter sequences, respectively. Stringent filtering revealed 78 genes in which the promoter/upstream region (−10 kb to +0.5 kb) was recognized by STAT5 both in WT and STAT5 overexpressing MEFs and 347 genes that bound STAT5 only in overexpressing cells. Genome-wide expression analyses identified that the majority of STAT5-bound genes was not under GH control. Up to 40% of STAT5-bound genes were not expressed. For the first time we demonstrate the magnitude of opportunistic genomic STAT5 binding that does not translate into transcriptional activation of neighboring genes.</P>