scholarly journals Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

2021 ◽  
Author(s):  
Weizheng Liang ◽  
Guipeng Li ◽  
Huanhuan Cui ◽  
Yukai Wang ◽  
Wencheng Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Evolutionary Analysis ◽  
Protein Sequence Analysis ◽  
Functional Differences ◽  
Transcriptional Effect

Abstract Background: Differences in gene expression, which arises from divergence in cis-regulatory elements or alterations in transcription factors (TFs) binding specificity, are one of the most important causes of phenotypic diversity during evolution. On one hand, changes in the cis-elements located in the vicinity of target genes affect TF binding and/or local chromatin environment, thereby modulating gene expression in one-to-one manner. On the other hand, alterations in trans-factors influence the expression of their target genes in a more pleiotropic fashion. Although evolution of amino acid sequences is much slower than that of non-coding regulatory elements, particularly for the TF DNA binding domains (DBD)， it is still possible that changes in TF-DBD might have the potential to drive large phenotypic changes if the resulting effects have a net positive effect on the organism’s fitness. If so, species-specific changes in TF-DBD might be positively selected. So far, however, this possibility has been largely unexplored.Results: By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.Conclusions: There were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Moreover, Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.

scholarly journals Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

2021 ◽  
Author(s):  
Weizheng Liang ◽  
Guipeng Li ◽  
Huanhuan Cui ◽  
Yukai Wang ◽  
Wencheng Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Phenotypic Diversity ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Evolutionary Analysis ◽  
Protein Sequence Analysis ◽  
Systematic Analysis

AbstractDifferences in gene expression, which can arise from divergence in cis-regulatory elements or alterations in transcription factors binding specificity, are one of the most important causes of phenotypic diversity during evolution. By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.Author summaryOur study 1) represented a first systematic analysis of species-specific adaptation in DNA binding pattern of transcription factor. Although the mouse-specific amino acid changes did not manifest functional impact in our system, several explanations may account for it (See Discussion part for the detail); 2) represented a first study of cis-regulation between two reproductively isolated species by using a novel allodiploid system; 3) demonstrated a higher conservation of transcriptional output than that of DNA binding, suggesting the evolvability/plasticity of the latter; 4) finally provided a rich data resource for Cdx2 mediated regulation, including gene expression, chromatin accessibility and DNA binding etc.

scholarly journals t(8;21)(q22;q22) fusion proteins preferentially bind to duplicated AML1/RUNX1 DNA-binding sequences to differentially regulate gene expression

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1392-1401 ◽  
Author(s):  
Akiko J. Okumura ◽  
Luke F. Peterson ◽  
Fumihiko Okumura ◽  
Anita Boyapati ◽  
Dong-Er Zhang
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Fusion Proteins ◽  
Target Genes ◽  
Regulatory Elements ◽  
Chromosome Abnormalities ◽  
Cancer Development ◽  
Dna Binding Site

AbstractChromosome abnormalities are frequently associated with cancer development. The 8;21(q22;q22) chromosomal translocation is one of the most common chromosome abnormalities identified in leukemia. It generates fusion proteins between AML1 and ETO. Since AML1 is a well-defined DNA-binding protein, AML1-ETO fusion proteins have been recognized as DNA-binding proteins interacting with the same consensus DNA-binding site as AML1. The alteration of AML1 target gene expression due to the presence of AML1-ETO is related to the development of leukemia. Here, using a 25-bp random double-stranded oligonucleotide library and a polymerase chain reaction (PCR)-based DNA-binding site screen, we show that compared with native AML1, AML1-ETO fusion proteins preferentially bind to DNA sequences with duplicated AML1 consensus sites. This finding is further confirmed by both in vitro and in vivo DNA-protein interaction assays. These results suggest that AML1-ETO fusion proteins have a selective preference for certain AML1 target genes that contain multimerized AML1 consensus sites in their regulatory elements. Such selected regulation provides an important molecular mechanism for the dysregulation of gene expression during cancer development.

scholarly journals Zinc finger protein SALL4 functions through an AT-rich motif to regulate gene expression

2020 ◽  
Author(s):  
Nikki R. Kong ◽  
Mahmoud A. Bassal ◽  
Hong Kee Tan ◽  
Jesse V. Kurland ◽  
Kol Jia Yong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Cancer Cells ◽  
Zinc Finger ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Embryonic Stem ◽  
Regulate Gene Expression ◽  
Protein Binding Microarray ◽  
Regulate Gene

SummaryThe zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, down-regulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive target for designing therapeutic strategies. However, whether SALL4 binds DNA directly to regulate gene expression is unclear and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) experiments, we identified and validated the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA-seq analyses after SALL4 knockdown, we discovered hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of Histone 3 Lysine 9-specific Demethylases (KDMs). Taken together, these results elucidated the mechanism of SALL4 DNA binding and revealed novel pathways and molecules to target in SALL4-dependent tumors.

scholarly journals The Gene for the Embryonic Stem Cell Coactivator UTF1 Carries a Regulatory Element Which Selectively Interacts with a Complex Composed of Oct-3/4 and Sox-2

1999 ◽  
Vol 19 (8) ◽  
pp. 5453-5465 ◽  
Author(s):  
Masazumi Nishimoto ◽  
Akiko Fukushima ◽  
Akihiko Okuda ◽  
Masami Muramatsu
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Es Cells ◽  
Regulatory Element ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
General Mechanism ◽  
Octamer Motif ◽  
Biochemical Analyses ◽  
Binding Sequence

ABSTRACT UTF1 is a transcriptional coactivator which has recently been isolated and found to be expressed mainly in pluripotent embryonic stem (ES) cells (A. Okuda, A. Fukushima, M. Nishimoto, et al., EMBO J. 17:2019–2032, 1998). To gain insight into the regulatory network of gene expression in ES cells, we have characterized the regulatory elements governing UTF1 gene expression. The results indicate that the UTF1 gene is one of the target genes of an embryonic octamer binding transcription factor, Oct-3/4. UTF1 expression is, like the FGF-4 gene, regulated by the synergistic action of Oct-3/4 and another embryonic factor, Sox-2, implying that the requirement for Sox-2 by Oct-3/4 is not limited to the FGF-4 enhancer but is rather a general mechanism of activation for Oct-3/4. Our biochemical analyses, however, also reveal one distinct difference between these two regulatory elements: unlike the FGF-4 enhancer, the UTF1 regulatory element can, by its one-base difference from the canonical octamer-binding sequence, selectively recruit the complex comprising Oct-3/4 and Sox-2 and preclude the binding of the transcriptionally inactive complex containing Oct-1 or Oct-6. Furthermore, our analyses reveal that these properties are dictated by the unique ability of the Oct-3/4 POU-homeodomain that recognizes a variant of the Octamer motif in the UTF1 regulatory element.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

scholarly journals Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal

2005 ◽  
Vol 280 (16) ◽  
pp. 16484-16498 ◽  
Author(s):  
Eduardo Martinez-Ceballos ◽  
Pierre Chambon ◽  
Lorraine J. Gudas
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Retinoic Acid ◽  
Leukemia Inhibitory Factor ◽  
Target Genes ◽  
Hox Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Inhibitory Factor ◽  
Hoxa Cluster

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of theHoxa1gene, the most 3′ member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1-/-embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1-/-mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1-/-ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1,Postn/Osf2, and the bone sialoprotein gene orBSP), genes that are expressed in the developing brain (e.g. Nnat,Wnt3a,BDNF,RhoB, andGbx2), and genes involved in various cellular processes (e.g. M-RAS,Sox17,Cdkn2b,LamA1,Col4a1,Foxa2,Foxq1,Klf5, andIgf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1,Oct3/4,Fgf4, andBmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1-/-ES cells express high levels of various endodermal markers, includingGata4andDab2, and express much lessFgf5after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.

scholarly journals HCR-FlowFISH: A flexible CRISPR screening method to identify cis-regulatory elements and their target genes

2020 ◽  
Author(s):  
SK Reilly ◽  
SJ Gosai ◽  
A Gutierrez ◽  
JC Ulirsch ◽  
M Kanai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Screening Method ◽  
Cell Types ◽  
Regulatory Elements ◽  
Hybridization Chain Reaction ◽  
Genome Wide ◽  
Wide Range ◽  
Causal Variants ◽  
Endogenous Loci

AbstractCRISPR screens for cis-regulatory elements (CREs) have shown unprecedented power to endogenously characterize the non-coding genome. To characterize CREs we developed HCR-FlowFISH (Hybridization Chain Reaction Fluorescent In-Situ Hybridization coupled with Flow Cytometry), which directly quantifies native transcripts within their endogenous loci following CRISPR perturbations of regulatory elements, eliminating the need for restrictive phenotypic assays such as growth or transcript-tagging. HCR-FlowFISH accurately quantifies gene expression across a wide range of transcript levels and cell types. We also developed CASA (CRISPR Activity Screen Analysis), a hierarchical Bayesian model to identify and quantify CRE activity. Using >270,000 perturbations, we identified CREs for GATA1, HDAC6, ERP29, LMO2, MEF2C, CD164, NMU, FEN1 and the FADS gene cluster. Our methods detect subtle gene expression changes and identify CREs regulating multiple genes, sometimes at different magnitudes and directions. We demonstrate the power of HCR-FlowFISH to parse genome-wide association signals by nominating causal variants and target genes.

scholarly journals Runx proteins regulate Foxp3 expression

2009 ◽  
Vol 206 (11) ◽  
pp. 2329-2337 ◽  
Author(s):  
Ludovica Bruno ◽  
Luca Mazzarella ◽  
Maarten Hoogenkamp ◽  
Arnulf Hertweck ◽  
Bradley S. Cobb ◽  
...  
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cd4 T Cells ◽  
Target Genes ◽  
De Novo ◽  
Foxp3 Expression ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Downstream Target ◽  
Runx Proteins

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes.

New insights into promoter–enhancer communication mechanisms revealed by dynamic single-molecule imaging

2021 ◽  
Author(s):  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Target Genes ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Single Molecule Imaging ◽  
Cell Type ◽  
Regulatory Information ◽  
Cell Type Specific ◽  
Target Promoters

Establishing cell-type-specific gene expression programs relies on the action of distal enhancers, cis-regulatory elements that can activate target genes over large genomic distances — up to Mega-bases away. How distal enhancers physically relay regulatory information to target promoters has remained a mystery. Here, we review the latest developments and insights into promoter–enhancer communication mechanisms revealed by live-cell, real-time single-molecule imaging approaches.

scholarly journals The transcriptional activator hepatocyte nuclear factor 6 regulates liver gene expression.

1996 ◽  
Vol 16 (11) ◽  
pp. 6273-6284 ◽  
Author(s):  
U Samadani ◽  
R H Costa
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Dna Binding ◽  
Target Genes ◽  
Site Directed Mutagenesis ◽  
Epithelial Cell Line ◽  
Homeodomain Protein ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Fatty Acid Binding

The hepatocyte nuclear factor 3(alpha) (HNF-3(alpha)), -3(beta), and -3(gamma) proteins share homology in the winged-helix/fork head DNA binding domain and mediate hepatocyte-enriched transcription of numerous genes whose expression is necessary for organ function. In this work, we identify a liver-enriched transcription factor, HNF-6, which recognizes the -138 to -126 region of the HNF-3(beta) promoter and binds the original HNF-3 site of the transthyretin promoter (-94 to -106). We show that HNF-6 and HNF-3 possess different DNA binding specificities by competition and methylation interference studies and are immunologically distinct. Site-directed mutagenesis of the HNF-6 sites in the HNF-3(beta) and transthyretin promoters diminishes reporter gene expression, suggesting that HNF-6 activates transcription of these promoters. Using the HNF-6 binding sequence DHWATTGAYTWWD (where W = A or T, Y = T or C, H is not G, and D is not C) determined by sequence comparison and methylation interference, we predicted that HNF-6 will bind to 22 additional hepatocyte-enriched genes. Of these potential target genes, we selected seven of the HNF-6 binding sequences and demonstrated that they bind the HNF-6 protein. These include promoter sequences from alpha-2 urinary globulin, alpha-1 antitrypsin, cytochrome P-450 2C13, L-type 6-phosphofructo-2-kinase, mouse major urinary protein, tryptophan oxygenase, and alpha-fetoprotein genes. HNF-6 binding activity was also found in the intestinal epithelial cell line HT29, and potential HNF-6 binding sites were present in intestinal sucrase isomaltase, cdx-2 homeodomain protein, and intestinal fatty acid binding protein promoter regions. These studies suggest that HNF-6 may regulate hepatocyte-specific genes and may play a role in epithelial cell differentiation of gut endoderm via regulation of HNF-3(beta).

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