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PubMed2025

Systematic decoding of functional enhancer connectomes and risk variants in human glioma.

Genetic and epigenetic variations contribute to the progression of glioma, but the mechanisms underlying these effects, particularly for enhancer-associated genetic variations in non-coding regions, still remain unclear. Here we performed high-throughput CRISPR interference screening to identify pro-tumour enhancers in glioma cells. By integrating genome-wide H3K27ac HiChIP data, we identified the target genes of these pro-tumour enhancers and revealed the essential role of enhancer connectomes in promoting glioma progression. Through systematic analysis of enhancers carrying glioma risk-associated single-nucleotide polymorphisms (SNPs), we found that these SNPs can promote glioma progression through the enhancer connectome. Using CRISPR-Cas9-mediated enhancer interference and SNP editing, we demonstrated that glioma-specific enhancer carrying the risk SNP rs2297440 regulates SOX18 expression by specifically recruiting transcription factor MEIS1 binding, thereby contributing to glioma progression. Our study sheds light on the molecular mechanisms underlying glioma susceptibility and provides potential therapeutic targets to treat glioma.

Humans↗

PubMed2006

The immunoglobulin heavy-chain gene 3' enhancers deregulate bcl-2 promoter usage in t(14;18) lymphoma cells.

In t(14;18) lymphomas, bcl-2 is juxtaposed to the immunoglobulin heavy-chain gene (IgH), resulting in increased bcl-2 transcription and resistance to apoptosis. Regulatory elements of both the bcl-2 promoter and the IgH enhancers are believed to play a role in the increased expression of bcl-2 in t(14;18) lymphoma cells. In addition, transcription of the translocated bcl-2 allele is deregulated with activation of the normally minor bcl-2 P2 promoter. The mechanisms involved in the promoter shift from P1 to P2 are not known. We found that the murine IgH 3' enhancers increased bcl-2 P2 promoter activity in an episomal model of the translocation, and IgH enhancer region HS12 had the greatest effect. Quantitative chromatin immunoprecipitation (ChIP) assays revealed that localized histone H3 hyperacetylation of the P2 promoter was observed on the translocated allele in t(14;18) DHL-4 cells and also on the stably transfected bcl-2 promoter-IgH enhancer episomal construct. Analysis of the HS12 enhancer region revealed that a previously identified nuclear factor-kappaB (NF-kappaB) site and a previously uncharacterized downstream Cdx site, both of which are conserved in the human and murine IgH enhancers, were important for its enhancer activity and promoter activation. ChIP assays showed that C/EBPbeta bound to the HS12 Cdx site in vivo, and mutation of this site abrogated the binding of C/EBPbeta. Reduced expression of C/EBPbeta by transfection of small interfering RNA or interference with NF-kappaB activity decreased transcription from the bcl-2 promoters. These results demonstrate that the IgH 3' enhancers, particularly HS12, are important for the deregulation of bcl-2 promoter usage in t(14;18) lymphomas.

Acetylation↗

PubMed2001

Assessing a role for enhancer-blocking activity in gene regulation within the murine T-cell receptor alpha/delta locus.

Although situated close together within the T-cell receptor (TCR) alpha/delta locus, TCR delta and TCR alpha gene segments are controlled by two developmental stage-specific enhancers and are activated according to distinct developmental programmes. We previously used a stable transfection colony assay to identify an enhancer-blocking element, blocking element alpha/delta-1 (BEAD-1), between the TCR delta and alpha gene segments of the human TCR alpha/delta locus. We hypothesized that enhancer-blocking by BEAD-1 might be required to prevent the TCR delta enhancer from activating TCR alpha gene segment transcription and rearrangement at the double negative stage of thymocyte development. Here, we used a transfection approach to define partial enhancer-blocking activity in an analogous position of the murine TCR alpha/delta locus. To test the functional significance of this activity in vivo, we used gene targeting to delete the region from the endogenous locus. We found no perturbation of TCR delta and TCR alpha gene expression and rearrangement on targeted alleles, indicating that enhancer-blocking activity in this region is not required to maintain the developmentally distinct activation profiles of the two genes. We suggest that appropriate regulation may be achieved as a result of intrinsic biases in enhancer-promoter interactions or a developmental stage specificity to promoter function that is distinct from any additional specificity imposed by the enhancers themselves.

Animals↗

PubMed1991

The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements.

The tissue-specific developmental regulation of the alpha, beta, gamma and delta T-cell antigen receptor (TCR) genes is controlled by the corresponding distinct enhancers and their enhancer-binding proteins. To find a common TCR regulatory element, we have studied the ability of the newly described enhancer-binding protein Gata3 to bind to the sequence motif (A/T)GATA(G/A) shared between enhancer elements of all four TCR genes. Gata3 was shown in the chicken to be an enhancer-binding protein containing a zinc-finger domain. We have isolated a full-length human Gata3 cDNA and characterized its role in TCR gene regulation. Northern blot analysis showed that the Gata3 mRNA is present in T-cell, but not B-cell, macrophage or HeLa cell lines. The in vitro transcribed and translated Gata3 cDNA product bound specifically to the enhancer elements of at least three TCR genes containing the above consensus sequence. This binding specificity was consistent with the in vivo activity of the TCR enhancer-binding element in transient transfection assays. Hence, the Gata3 protein, together with other T-cell-specific enhancer-binding proteins, may form the basis of tissue-specific expression of the TCR genes.

Amino Acid Sequence↗

PubMed1993

p70 lupus autoantigen binds the enhancer of the T-cell receptor beta-chain gene.

The p70 (Ku) autoantigen has been described as a nonhistone nuclear protein recognized by antibodies from lupus patients. In our studies on the regulation of T-cell receptor (TCR) beta-chain gene expression we have identified the p70 lupus autoantigen as a DNA-binding protein that binds the enhancer of the TCR beta-chain gene. This enhancer is essential for expression of the TCR beta gene. The core TCR beta enhancer contains the E3 motif, which we show here is essential for enhancer activity. The protection of the E3 motif in T cells and the marked reduction in enhancer activity when the E3 motif is mutated underline its physiological importance in regulating beta enhancer activity. The p70 lupus autoantigen gene was identified by screening T-cell lambda gt11 libraries with an E3 probe. The gene encodes a protein which binds the E3 motif in a sequence-specific manner. The identification of a 70-kDa protein as a major E3-binding protein by UV crosslinking is consistent with the conclusion that the p70 lupus autoantigen binds the beta enhancer. Finally, we have shown that T-cell nuclear proteins which bind the E3 motif bear p70 (Ku) lupus autoantigenic determinants. Together these data suggest that the p70 autoantigen binds a critical motif in the beta enhancer and probably regulates TCR beta gene expression.

Animals↗

PubMed1997

Enhancer blocking activity located near the 3' end of the sea urchin early H2A histone gene.

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during development is regulated by gene-specific elements. To learn how within the histone repeating unit a gene-specific activator can be prevented to communicate with the heterologous promoters, we searched for domain boundaries by using the enhancer blocking assay. We focused on the region near the 3' end of the H2A gene where stage-specific nuclease cleavage sites appear upon silencing of the early histone genes. We demonstrated that a DNA fragment of 265 bp in length, defined as sns (for silencing nucleoprotein structure), blocked the enhancer activity of the H2A modulator in microinjected sea urchin embryos only when placed between the enhancer elements and the promoter. We also found that sns silenced the modulator elements even when placed at 2.7 kb from the promoter. By contrast, the enhancer activity of the modulator sequences, located downstream to the coding region, was not affected when sns was positioned in close proximity to the promoter. Finally, the H2A sns fragment placed between the simian virus 40 regulative region and the tk promoter repressed chloramphenicol acetyltransferase expression in transfected human cell lines. We conclude that 3' end of the H2A gene contains sequence elements that behave as functional barriers of enhancer function in the enhancer blocking assay. Furthermore, our results also indicate that the enhancer blocking function of sns lacks enhancer and species specificity and that it can act in transient assays.

Amino Acid Sequence↗

PubMed1995

Mutually exclusive interaction of a novel matrix attachment region binding protein and the NF-muNR enhancer repressor. Implications for regulation of immunoglobulin heavy chain expression.

The immunoglobulin heavy chain (IgH) intronic enhancer stimulates transcription from functional promoters in B lymphocytes but not other cell types. The observation that binding sites for the nuclear factor-mu negative regulator (NF-muNR) enhancer repressor overlap nuclear matrix attachment regions (MARs) in this enhancer has lead to the hypothesis that the cell type specificity of the enhancer might be controlled by regulating nuclear matrix attachment (Scheuermann, R. H., and Chen, U. (1989) Genes & Dev. 3, 1255-1266). To understand the role of MARs in IgH enhancer regulation, we have identified a novel MAR-binding protein, MAR-BP1, from soluble nuclear matrix preparations based on its ability to bind to the MARs associated with the IgH enhancer. Purified MAR-BP1 migrates as a 33-kDa protein, and it can be found in nuclear matrix preparations from a number of different types of lymphoid cell lines. Although specific binding sites have been difficult to localize by chemical or enzymatic footprinting procedures, NF-muNR binding sites are critical for efficient MAR-BP1 binding. Indeed, binding of the IgH enhancer to either intact nuclear matrix preparations or to MAR-BP1 is mutually exclusive to NF-muNR binding. These results are consistent with a model for cell-type specific regulation in which binding of the NF-muNR repressor to the IgH enhancer prevents nuclear matrix attachment in inappropriate cells by interfering with MAR-BP1/enhancer interaction.

Animals↗

PubMed1996

Purification and properties of rat liver nuclear proteins that interact with the hepatitis B virus enhancer 1.

The hepatitis B virus enhancer 1 element plays a fundamental role in the liver-specific regulation of hepatitis B virus gene expression. A central region of enhancer 1, the enhancer core domain, contains at least four cis-acting sequence motifs that are essential for enhancer 1 activity. In this study, we have investigated an essential motif within the core domain previously defined as footprint V (FPV). Transient transfection analyses demonstrate that FPV is capable of independently functioning in a liver-specific manner to activate transcription. Therefore, to further examine the liver-specific properties of FPV-mediated enhancer 1 activity, we have carried out the biochemical purification and characterization of FPV binding activity from rat liver nuclei. This study has conclusively identified hepatocyte nuclear factor 3beta (HNF-3beta), a liver-enriched member of the HNF-3/forkhead gene family, as the predominant purified protein that interacts with the FPV motif. Moreover, a cellular protein(s) that copurified with HNF-3beta specifically interacts with a novel sequence motif that partially overlaps FPV. Since this novel motif contains a palindromic sequence, we have tentatively referred to the protein(s) that binds to this site as palindrome-binding factor (PBF). Additional evidence indicates that HNF-3beta and PBF cooperatively interact with enhancer 1. Therefore, this study supports the hypothesis that FPV-mediated enhancer activity involves a cooperative interplay between HNF-3beta and at least one other enhancer 1-binding protein, PBF.

Activating Transcription Factor 2↗

PubMed1996

Identification of functional elements of the chicken epsilon-globin promoter involved in stage-specific interaction with the beta/epsilon enhancer.

Expression of the chicken globin genes is regulated in part by competition between the betaA-globin and epsilon-globin promoters for the enhancer found between the genes. To understand the determinants of the enhancer-promoter interaction in stage-specific regulation, the functional elements of the embryonic chicken epsilon-globin promoter were characterized. In vitro assays demonstrated that: (a) the TATA motif at -30 bound GATA-1, (b) Sp1 bound to an element centered at -54, and (c) both Sp1 and another factor, designated CACCC (which appears related to erythroid Krüppel-like factor, EKLF) bound in the -120 to -128 region. The functions of these motifs were tested using transient expression in embryonic erythroid cells. In the absence of the enhancer, promoter point mutants showed that the TATA, Sp1, and CCAAT motifs (but not the CACCC motif) contributed to promoter activity. In contrast, in the presence of the enhancer, all four motifs (including the CACCC motif) contributed to transcription. Developmental regulation of the enhancer activity was observed, with enhancement decreasing sharply from 185-fold at 4 days (cells expressing epsilon-globin) to 16-fold at 10 days (when epsilon-globin is no longer expressed). Taken together, the data suggest that multiple transcription factors contribute to promoter-enhancer interaction and the developmental regulation of epsilon-globin expression, with EKLF-like factors having an especially important role. Regulation of stage specificity occurs at the level of enhancer/epsilon-promoter interaction, even in the absence of competition, and is not simply a property of the enhancer or promoter in isolation.

Animals↗

PubMed1997

Identification of an upstream enhancer within a functional promoter of the human leukemia inhibitory factor receptor gene and its alternative promoter usage.

Knockout of the leukemia inhibitory factor receptor (LIFR) gene results in disrupted placental architecture, imbalanced bone development, and losses of functional neurons. We here report the identification of an enhancer in a functional human LIFR gene promoter and alternative promoter usage by this gene. A single transcription start site was identified in placental JEG-3 cells and a genomic clone containing 4876-nucleotide upstream sequences was found to have promoter activity in JEG-3 cells. However, in osteogenic sarcoma U-2 OS cell, Northern blot using a probe of the first exon detected in JEG-3 cells failed to detect LIFR transcripts. 5'-Rapid amplification of cDNA ends (RACE) revealed an alternative first exon and a 0.6-kilobase pair (kb) 5'-flanking region possessed promoter activity in U-2 OS cells. For the 4.8-kb promoter active in placental cells, a minimal promoter was localized within -162 nucleotides. Three regions increased and one inhibited promoter activity. Subcloning of an activation region (-4876 to -3453 nucleotides) into SV40 promoter either upstream or downstream in either orientation to the luciferase reporter resulted in 10-35-fold luciferase induction, demonstrating the characteristics of an enhancer. Transfections into nine cell lines of different tissue origin indicated that the cloned promoter and enhancer in the 4.8-kb fragment was placental tissue-specific. A 226-base pair fragment (-4625 to -4400 nucleotides) was further localized as the minimal enhancer region, in which deletion of either element A (-4625 to -4581 nucleotides) or element B (-4418 to -4400 nucleotides) resulted in the loss of enhancer activity. Electrophoretic mobility shift assay confirmed that these two elements bind to specific nuclear proteins individually. In the middle region between element A and B, disruption of enhancer integrity also led to a loss of enhancer activity, although two SP1 and three NF-kappaB/c-Rel binding sites did not contribute to enhancer function. These results demonstrate a complex regulation of the human LIFR gene, including alternative promoter usage and tissue-specific elements at the transcription level.

Base Sequence↗

PubMed1998

Mechanisms in the transcriptional regulation of bradykinin B1 receptor gene expression. Identification of a minimum cell-type specific enhancer.

To investigate the mechanisms of bradykinin B1 (BKB1) receptor gene expression, transient DNA transfection analyses of human BKB1 receptor gene promoter were performed in SV-40 transformed IMR90 cells. A positive regulatory element (PRE) located at position -604 to -448 base pair (bp) upstream of the transcription start site consistently exhibited, by far, the highest level of relative luciferase activity. A negative regulatory element, at position -682 to -604 bp, was able to completely ablate the function of the PRE. Transfection combined with deletion and mutation analyses illustrated that the PRE contains a classic, powerful enhancer. This enhancer was minimized to a 100-bp element at position -548 to -448 bp. A 78-bp fragment of negative regulatory element functioned as a silencer. Transient transfection of the enhancer construct, driven by heterologous herpes simplex thymidine kinase promoter, into a variety of cell types, showed that this enhancer presents a cell-type specific feature. In the characterization of the enhancer, motifs A (-548 to -532) and B (-483 to -477) were found to be essential for full enhancer activity. Motif D (-472 to -467) played a smaller role in enhancer activation. Gel shift and antibody supershift assays determined that an AP-1 factor binds with motif B. The nuclear protein which binds to motif A has yet to be identified. Both factors are the critical regulators for this enhancer activation.

Animals↗

PubMed2000

PU.1-mediated transcription is enhanced by HMG-I(Y)-dependent structural mechanisms.

The ets transcription factor PU.1 is an important regulator of the immunoglobulin heavy chain gene intronic enhancer, or mu enhancer. However, PU.1 is only one component of the large multiprotein complex required for B cell-specific enhancer activation. The transcriptional coactivator HMG-I(Y), a protein demonstrated to physically interact with PU.1, increases PU.1 affinity for the mu enhancer muB element, indicating that HMG-I(Y) may play a role in the transcriptionally active mu enhanceosome. Increased PU.1 affinity is not mediated by HMG-I(Y)-induced changes in DNA structure. Investigation of alternative mechanisms to explain the HMG-I(Y)-mediated increase in PU.1/mu enhancer binding demonstrated, by trypsin and chymotrypsin mapping, that interaction between PU.1 and HMG-I(Y) in solution induces a structural change in PU.1. In the presence of HMG-I(Y) and wild-type mu enhancer DNA, PU.1 becomes more chymotrypsin resistant, suggesting an additional change in PU.1 structure upon HMG-I(Y)-induced PU.1/DNA binding. From these results, we suggest that increased DNA affinity under limiting PU.1 concentrations is mediated by an HMG-I(Y)-induced structural change in PU.1. In functional assays, HMG-I(Y) further augments transcriptional synergy between PU.1 and another member of the ets family, Ets-1, indicating that HMG-I(Y) is a functional component of the active enhancer complex. These studies suggest a new mechanism for HMG-I(Y)-mediated coactivation; HMG-I(Y) forms protein-protein interactions with a transcription factor, which alters the three-dimensional structure of the factor, resulting in enhanced DNA binding and transcriptional activation. This mechanism may be important for transcriptional activation under conditions of limiting transcription factor concentration, such as at the low levels of PU.1 expressed in B cells.

3T3 Cells↗

PubMed2002

Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor beta1 (TGF-beta1) and accompanied by transient activation of the vascular smooth muscle alpha-actin (SmalphaA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmalphaA enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-beta1. However, TGF-beta1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmalphaA enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate SmalphaA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmalphaA enhancer required for TGF-beta1 amplification. Additionally, the TGF-beta1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmalphaA enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the SmalphaA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmalphaA enhancer provides new insight into the poorly understood process of myofibroblast differentiation.

Actins↗

PubMed1998

Mechanism of contrast enhancement in breast lesions at MR imaging.

PURPOSE: The aim of the study was to explain why breast lesions are enhanced by contrast medium at MR imaging and to elucidate the histopathological basis for the overlap in the enhancement patterns of benign and malignant breast lesions. MATERIAL AND METHODS: Ten invasive breast carcinomas and 10 benign breast lesions were selected for the study. Of the 10 carcinomas, 5 showed a strong and early contrast enhancement, and 5 did not. Of the 10 benign lesions, 5 showed a strong and early contrast enhancement, and 5 showed no enhancement. The following morphometric variables were evaluated: proliferation cell index of neoplastic parenchymal cells, S-phase fraction, nuclear ploidy analysed by image DNA-cytometry, microvessel density, and the percentage proportion of the interstitial area. RESULTS: Contrast enhancement was related to the proliferating activity of the hyperplastic or neoplastic parenchymal cells and was inversely correlated with the interstitial area in carcinomas as well as in benign tumours and non-neoplastic lesions of the breast. CONCLUSION: Morphometric variables play an important role in the general mechanism of MR contrast enhancement in examinations of the breast and explain the histopathological basis for the overlap in the enhancement patterns of benign and malignant breast lesions.

Breast↗

PubMed1990

A family of homologous X chromosomal sequences with some members showing evidence of enhancer activity.

Little is known about either the frequency of occurrence of enhancers or the interrelationships of various enhancers in mammalian genomes. Previous reports described a potent, transcriptional enhancer within the sequence termed "Xrep," a human X chromosomal genomic clone (Riley et al., 1986; Riley, 1989a). Additional Xrep-related sequences reside on the human X chromosome. A probe for the Xrep enhancer hybridized with multiple restriction fragments located on the X chromosome. Two of the fragments cross-hybridizing with the enhancer had identical enhancer-like sequences but dissimilar flanking sequences. However, both of the sequenced enhancers were flanked by poly(GT) repeats strongly implicated in the generation of polymorphisms. A third sequence cross-hybridizing to the enhancer probe was found, in chromatin form, to be extremely DNase I hypersensitive.

Animals↗

PubMed1991

Enhancer, repressor, and promoter specificities combine to regulate the rat alpha-fetoprotein gene.

The upstream transcription control region of the rat alpha-fetoprotein (AFP) gene was analyzed using transient expression of CAT genes in HepG2 cells which express the gene; H4C3 cells which repress the AFP gene but express the albumin gene; and four nonexpressing cell lines. Deletion analysis based on the DNA sequence resolved three upstream enhancers corresponding to the mouse AFP enhancers, but showed additional weak effects from flanking sequences. Quantitative experiments demonstrated that the three enhancers were additive when acting through a single promoter and did not confirm the presence of a distal upstream repressor. All three enhancers stimulated the AFP, albumin, or thymidine kinase (tk) promoter in HepG2, but only the tk and albumin promoters in H4C3. Deletion of a proximal repressor region near the AFP promoter allowed expression in H4C3 cells with the AFP promoter. Thus, the liver-specific developmental repressor is near the AFP promoter, and H4C3 cells provide an in vitro system for analysis of this repressor in transfection assays. The repressor region also blocked expression of the SV40 enhancer through the AFP promoter in hepatic and nonhepatic cell lines, but when this enhancer was combined with an AFP promoter from which the repressor region was deleted, the combination showed expression in all six cell lines studied. AFP expression results from a combination of enhancer, promoter, and repressor activities, and the repressor is functional with a heterologous enhancer in a variety of cells.

Animals↗

PubMed2006

Evidence of the capability of the CMV enhancer to activate in trans gene expression in mammalian cells.

Studies have reported that an enhancer can act in trans when artificially, noncovalently bridged to the promoter by a protein-linked biotin:streptavidin complex, or when an enhancer and a promoter are located on separate concatenated plasmids. To investigate such transactivation in mammalian cells, we constructed CMV promoter-enhancer mutants driving the expression of the EGFP reporter gene and transfected cultured cells with various combinations of the mutant PCR products; results were analyzed using fluorescence microscopy and flow cytometry. Our results show that the CMV enhancer can stimulate transcription in trans, even in the absence of physical association of the enhancer and promoter. Furthermore, we show that the transactivation of the CMV enhancer can be strengthened by the histone deacetylase inhibitor sodium butyrate. Finally, we provide evidence that the CMV enhancer can influence, in trans, the activity of heterologous promoters. Although different mechanisms may lead to transcriptional activation when the CMV enhancer is not covalently linked to the promoter, our results suggest that the main mechanism resembles the process of transfection and may be important for gene regulation. These findings may have implications in understanding the processes that underlie gene therapy because of the potential alteration of endogenous gene expression.

Animals↗

PubMed1999

Stimulation of Tat-associated kinase-independent transcriptional elongation from the human immunodeficiency virus type-1 long terminal repeat by a cellular enhancer.

The human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) initiates transcription efficiently but produces only short transcripts in the absence of the trans-activator protein, Tat. To determine whether a cellular enhancer could provide the signals required to recruit an elongation-competent polymerase to the HIV-1 LTR, the B cell-specific immunoglobulin heavy chain gene enhancer (IgHE) was inserted upstream of the LTR. The enhancer increased transcription in the absence of Tat between 6- and 7-fold in transfected B cells, but the full-length transcripts remained at basal levels in HeLa cells, where the enhancer is inactive. RNase-protection studies showed that initiation levels in the presence and absence of the enhancer were constant, but the enhancer significantly increased the elongation capacity of the polymerases. Tat-stimulated elongation is strongly inhibited by the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which inhibits the Tat-associated kinase, TAK (CDK9). However, polymerases initiating transcription from LTRs carrying the enhancer were able to efficiently elongate in the presence of DRB. Specific repression of TAK by expression in trans of the CDK9 kinase also inhibited Tat-stimulated elongation but did not inhibit enhancer-dependent transcription significantly. Thus, the activation of polymerase processivity by the IgHE involves a unique mechanism which is independent of TAK.

B-Lymphocytes↗