Yugong Ho

A Defined Locus Control Region Determinant Links Chromatin Domain Acetylation with Long-Range Gene Activation

Gene activation in higher eukaryotes is often under the control of regulatory elements quite distant from their target promoters. It is unclear how such long-range control is mediated. Here we show that a single determinant of the human growth hormone locus control region (hGH LCR) located 14.5 kb 5prime prime or minute to the hGH-N promoter has a critical, specific, and nonredundant role in facilitating promoter trans factor binding and activating hGH-N transcription. Significantly, this same determinant plays an essential role in establishing a 32 kb acetylated domain that encompasses the entire hGH LCR and the contiguous hGH-N promoter. These data support a model for long-range gene activation via LCR-mediated targeting and extensive spreading of core histone acetylation.

Long-range enhancer activity determines Myc sensitivity to Notch inhibitors in T cell leukemia

Significance The protooncogene c-Myc (Myc) is an oncogenic driver in many cancers, but is difficult to target directly with drugs. An alternative strategy is to use drugs that inhibit factors that regulate Myc expression. Notch drives Myc expression in most T-cell leukemias, but clinical trials of Notch inhibitors have been disappointing, possibly because cells emerge that express Myc in a Notch-independent fashion. Here we identify the genomic switches that regulate Myc expression in the Notch-inhibitor–sensitive and –resistant states. Our findings suggest that Notch inhibitor resistance occurs through a “switch swap” that relieves Notch dependency while increasing dependency on a different factor, bromodomain containing 4 (Brd4). These studies provide a rationale for targeting Myc in T cell leukemias with combinations of Notch and Brd4 inhibitors. Notch is needed for T-cell development and is a common oncogenic driver in T-cell acute lymphoblastic leukemia. The protooncogene c-Myc (Myc) is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates Myc is unknown. Here, we identify a distal enhancer located >1 Mb 3′ of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter. The Notch1 binding element in this region activates reporter genes in a Notch-dependent, cell-context–specific fashion that requires a conserved Notch complex binding site. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Leukemia cells selected for resistance to Notch inhibitors express Myc despite epigenetic silencing of enhancer domains near the Notch transcription complex binding sites. Notch-independent expression of Myc in resistant cells is highly sensitive to inhibitors of bromodomain containing 4 (Brd4), a change in drug sensitivity that is accompanied by preferential association of the Myc promoter with more 3′ enhancer domains that are strongly dependent on Brd4 for function. These findings indicate that altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia.

The juxtaposition of a promoter with a locus control region transcriptional domain activates gene expression

Nonlinear chromatin configurations can juxtapose widely separated elements within a genomic locus; however, it remains unclear how these structures are established and contribute to transcriptional control. A 5′‐remote locus control region (LCR) regulates the human growth hormone (hGH‐N) gene. HSI, a pituitary‐specific component of the hGH LCR, establishes a domain of polymerase II (PolII) transcription 5′ to hGH‐N. Repression of this transcriptional domain by HSI deletion or PolII blockade decreases hGH‐N expression. Here, we show that hGH‐N activation is accompanied by positioning of the hGH‐N promoter to this LCR transcriptional domain. Selectively blocking LCR transcription inhibits the formation of this active ‘looped’ conformation. Thus, HSI is crucial for establishing a domain of noncoding PolII transcription, and this domain is intimately linked with chromatin organization of the active hGH‐N locus. This integration of LCR transcription with chromatin reconfiguration constitutes a robust pathway for long‐range gene activation.

A single base difference between Pit-1 binding sites at the hGH promoter and locus control region specifies distinct Pit-1 conformations and functions

ABSTRACT Activation of the human growth hormone (hGH-N) gene in pituitary somatotropes is mediated by a locus control region (LCR). This LCR is composed of DNase I-hypersensitive sites (HS) located −14.5 kb to −32 kb relative to the hGH-N promoter. HSI, at −14.5 kb, is the dominant determinant of hGH-N expression and is essential for establishment of a 32-kb domain of histone acetylation that encompasses the active hGH locus. This activity is conferred by three binding sites for the POU domain transcription factor Pit-1. These Pit-1 elements are sufficient to activate hGH-N expression in the mouse pituitary. In contrast, Pit-1 sites at the hGH-N promoter are consistently unable to mediate similar activity. In the present study, we demonstrate that the functional difference between the promoter-proximal and the HSI Pit-1 binding sites can be attributed in part to a single base difference. This base affects the conformation of the Pit-1/DNA complex, and reciprocal exchange of the divergent bases between the two sets of Pit-1 elements results in a partial reversal of their transgenic activities. These data support a model in which the Pit-1 binding sites in the hGH LCR allosterically program the bound Pit-1 complex for chromatin activating functions.

Distinct Chromatin Configurations Regulate the Initiation and the Maintenance of hGH Gene Expression

ABSTRACT For many mammalian genes, initiation of transcription during embryonic development must be subsequently sustained over extensive periods of adult life. It remains unclear whether maintenance of gene expression reflects the same set of pathways as are involved in initial gene activation. The human pituitary growth hormone (hGH-N) locus is activated in the differentiating somatotrope midway through embryogenesis by a multicomponent locus control region (LCR). DNase I-hypersensitive site I (HSI) of the LCR is essential to full developmental activation of the hGH-N locus. Here we demonstrate that conditional deletion of HSI from the active hGH locus in the adult pituitary effectively silences hGH-N expression. Analyses of chromatin structure and locus positioning demonstrate that a specific subset of the HSI functions active in the embryo retain their HSI dependence in the adult pituitary. These functions sustain engagement of the hGH locus with polymerase II (Pol II) factories, histone acetylation at the hGH-N promoter, and looping of the LCR to its target promoter. These data reveal that HSI is essential to both the maintenance and the initiation phases of gene expression. These observations contribute to our mechanistic understanding of how stable patterns of mammalian gene expression are established in a terminally differentiated cell.

DNase I Hypersensitive Site II of the Human Growth Hormone Locus Control Region Mediates an Essential and Distinct Long-range Enhancer Function

Background: A series of distal DNA elements regulates the human growth hormone gene. Results: Deletion of one of these elements, HSII, results in reduced growth hormone expression and changes in several activating phenomena at the locus. Conclusion: HSII is a unique regulator of growth hormone expression. Significance: Elucidating complex regulation determinants is imperative to the understanding of metazoan gene expression. Locus control regions (LCRs) comprise sets of DNA elements capable of establishing autonomous chromatin domains that support robust and physiologically appropriate expression of target genes, often working over extensive distances. Human growth hormone (hGH-N) expression in the pituitary is under the regulation of a well characterized LCR containing four DNase I hypersensitive sites (HSs). The two pituitary-specific HS, HSI and HSII, are located 14.5 and 15.5 kb 5′ to the hGH-N promoter. HSI is essential for activation of hGH-N during pituitary development and for sustaining robust activity in the adult. To determine whether the closely linked HSII has a role in hGH-N expression, it was deleted from a previously validated hGH/P1 transgene. Analysis of three independent hGH/P1(ΔHSII) transgenic mouse lines revealed that this deletion had no adverse effect on the formation of HSI, yet resulted in a substantial loss (70%) in hGH-N mRNA expression. This loss of expression was accompanied by a corresponding reduction in recruitment of the pituitary-specific transcription factor Pit-1 to the hGH-N promoter and a selective decrease in promoter occupancy of the elongation-linked isoform of RNA polymerase II. Sufficiency of HSI and HSII in LCR activity was explored by establishing two additional sets of mouse transgenic lines in which DNA segments containing these HS were positioned within the λ phage genome. In this “neutral” DNA context, HSII was required for the recruitment of HAT activity. These data establish HSII as a nonredundant component of the hGH LCR essential for establishment of robust levels of hGH-N gene expression.

The Role of the hGH Locus Control Region in Somatotrope Restriction of hGH-N Gene Expression

Expression of mammalian GH is normally restricted to somatotropes and somatolactotropes (somatotrope lineages) in the anterior pituitary. The basis for this restriction remains incompletely understood. Recent studies indicate that deoxyribonuclease I hypersensitive site I (HSI) of the hGH locus control region, located at -14.5 kb relative to the hGH-N promoter, acts as a potent long-range enhancer of hGH-N transcription. Here we report that HSI is also critical to somatotrope-restriction of hGH-N expression. Loss of HSI activity, either by direct inactivation of HSI or by interference with HSI-dependent downstream events, results in a relaxation of hGH-N cell-type specification with expansion of hGH-N expression to the full spectrum of Pit-1 positive pituitary cell types. These findings expand the defined roles for HSI of the hGH locus control region to include somatotrope lineage restriction as well as transcriptional enhancement of hGH-N gene expression.

Research Resource: T-Antigen Transformation of Pituitary Cells Captures Three Novel Cell Lines in the Pit-1 Lineage

We report the establishment of three distinct pituitary-derived murine cell lines generated by targeted T-antigen-induced transformation. The Pit1/0 line expresses pituitary-specific transcription factor-1 (Pit-1) but lacks expression of GH, prolactin (Prl), or TSH, and the Pit1/Prl line is selectively positive for Pit-1 and Prl. The third line, Pit1/Triple, expresses Pit-1 and all three of the Pit-1-dependent hormones: GH, Prl, and TSHβ/glycoprotein hormone α-subunit. The three corresponding transformation events appear to have captured pituitary cells representing: 1) an initial step in the Pit-1(+) lineage, 2) a cell line that corresponds to the differentiated lactotrope, and 3) a novel tri-hormone intermediate that may represent a pivotal step in Pit-1(+) cell lineage differentiation. The documented dependence of the tri-hormone expression in the Pit-1/Triple line on Pit-1 activity supports its potential role in the pathway of pituitary cell differentiation. The presence of a 123-kb human transgene encompassing the hGH locus (hGH/bacterial artificial chromosome) in two of these lines, Pit1/0 and Pit1/Prl, further expands their potential utility to the analysis of gene activation within the hGH gene cluster.

An Autoregulatory Pathway Establishes the Definitive Chromatin Conformation at the Pit-1 Locus

ABSTRACT The transcription factor Pit-1 (POU1-F1) plays a dominant role in cell lineage expansion and differentiation in the anterior pituitary. Prior studies of the mouse Pit-1 (mPit-1) gene revealed that this master regulatory locus is activated at embryonic day 13.5 (E13.5) by an early enhancer (EE), whereas its subsequent expression throughout adult life is maintained by a more distal definitive enhancer (DE). Here, we demonstrate that the sequential actions of these two enhancers are linked to corresponding shifts in their proximities to the Pit-1 promoter. We further demonstrate that the looping of the definitive enhancer to the mPit-1 promoter is critically dependent on a self-sustaining autoregulatory mechanism mediated by the Pit-1 protein. These Pit-1-dependent actions are accompanied by localized recruitment of CBP and enrichment for H3K27 acetylation within the Pit-1 locus. These data support a model in which the sequential actions of two developmentally activated enhancers are linked to a corresponding shift in higher-order chromatin structures. This shift establishes an autoregulatory circuit that maintains durable expression of Pit-1 throughout adult life.

Transcriptome analyses of female somatotropes and lactotropes reveal novel regulators of cell identity in the pituitary

The differentiation of the hormone-producing cell lineages of the anterior pituitary represents an informative model of mammalian cell fate determination. The generation and maintenance of two of these lineages, the GH-producing somatotropes and prolactin (PRL)-producing lactotropes, are dependent on the pituitary-specific transcription factor POU1F1. Whereas POU1F1 is expressed in both cell types, and plays a direct role in the activation of both the Gh and Prl genes, GH expression is restricted to somatotropes and PRL expression is restricted to lactotropes. These observations imply the existence of additional, cell type-enriched factors that contribute to the somatotrope and lactotrope cell identities. In this study, we use transgenic mouse models to facilitate sorting of somatotrope and lactotrope populations based on the expression of fluorescent markers expressed under Gh and Prl gene transcriptional controls. The transcriptomic analyses reveal a concordance of gene expression profiles in the two populations. The limited number of divergent mRNAs between the two populations includes a set of transcription factors that may have roles in pituitary lineage divergence and/or in regulating expression of cell type-specific genes after differentiation. Four of these factors were validated for lineage enrichment at the level of protein expression, two somatotrope enriched and two lactotrope enriched. Three of these four factors were shown to have corresponding activities in appropriate enhancement or repression of landmark genes in a cell culture model system. These studies identify novel regulators of the somatotropes and lactotropes, and they establish a useful database for further study of these lineages in the anterior pituitary.