Dynamic Interactions between Pit-1 and C/EBPα in the Pituitary Cell Nucleus

ABSTRACT The homeodomain (HD) transcription factors are a structurally conserved family of proteins that, through networks of interactions with other nuclear proteins, control patterns of gene expression during development. For example, the network interactions of the pituitary-specific HD protein Pit-1 control the development of anterior pituitary cells and regulate the expression of the hormone products in the adult cells. Inactivating mutations in Pit-1 disrupt these processes, giving rise to the syndrome of combined pituitary hormone deficiency. Pit-1 interacts with CCAAT/enhancer-binding protein alpha (C/EBPα) to regulate prolactin transcription. Here, we used the combination of biochemical analysis and live-cell microscopy to show that two different point mutations in Pit-1, which disrupted distinct activities, affected the dynamic interactions between Pit-1 and C/EBPα in different ways. The results showed that the first α-helix of the POU-S domain is critical for the assembly of Pit-1 with C/EBPα, and they showed that DNA-binding activity conferred by the HD is critical for the final intranuclear positioning of the metastable complex. This likely reflects more general mechanisms that govern cell-type-specific transcriptional control, and the results from the analysis of the point mutations could indicate an important link between the mislocalization of transcriptional complexes and disease processes.

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A mutation of the β-domain in POU1F1 causes pituitary deficiency due to dominant PIT-1β expression

Acta Endocrinologica ◽

10.1530/eje-20-1313 ◽

2021 ◽

Author(s):

Shigeru Suzuki ◽

Kumihiro Matsuo ◽

Yoshiya Ito ◽

Atsushi Kobayashi ◽

Takahide Kokumai ◽

...

Keyword(s):

Hormone Deficiency ◽

Luciferase Reporter ◽

Heterozygous Mutation ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Transactivation Domain ◽

Exon 2 ◽

Pituitary Development ◽

Long Term Follow Up ◽

Alternatively Spliced

Background: POU1F1 encodes both PIT-1α, which plays pivotal roles in pituitary development and GH, PRL and TSHB expression, and the alternatively spliced isoform PIT-1β, which contains an insertion of 26-amino acids (β-domain) in the transactivation domain of PIT-1α due to the use of an alternative splice acceptor at the end of the first intron. PIT-1β is expressed at much lower levels than PIT-1α and represses endogenous PIT-1α transcriptional activity. Although POU1F1 mutations lead to combined pituitary hormone deficiency (CPHD), no patients with β-domain mutations have been reported. Results: Here, we report that a three-generation family exhibited different degrees of CPHD, including growth hormone deficiency with intrafamilial variability of prolactin/TSH insufficiency and unexpected prolactinoma occurrence. The CPHD was due to a novel POU1F1 heterozygous variant (c.143-69T>G) in intron 1 of PIT-1α (RefSeq number NM_000306) or as c.152T>G (p.Ile51Ser) in exon 2 of PIT-1β (NM_001122757). Gene splicing experiments showed that this mutation yielded the PIT-1β transcript without other transcripts. Lymphocyte PIT-1β mRNA expression was significantly higher in the patients with the heterozygous mutation than a control. A luciferase reporter assay revealed that the PIT-1β-Ile51Ser mutant repressed PIT-1α and abolished transactivation capacity for the rat prolactin promoter in GH3 pituitary cells. Conclusions: We describe, for the first time, that PIT-1β mutation can cause CPHD through a novel genetic mechanism, such as PIT-1β overexpression, and that POU1F1 mutation might be associated with a prolactinoma. Analysis of new patients and long-term follow-up are needed to clarify the characteristics of PIT-1β mutations.

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Lhx4 and Prop1 are required for cell survival and expansion of the pituitary primordia

Development ◽

10.1242/dev.129.18.4229 ◽

2002 ◽

Vol 129 (18) ◽

pp. 4229-4239 ◽

Cited By ~ 1

Author(s):

Lori T. Raetzman ◽

Robert Ward ◽

Sally A. Camper

Keyword(s):

Cell Survival ◽

Anterior Pituitary ◽

Molecular Genetic ◽

Cell Types ◽

Hormone Deficiency ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Temporal Shift ◽

Pituitary Development ◽

Show Evidence

Deficiencies in the homeobox transcription factors LHX4 and PROP1 cause pituitary hormone deficiency in both humans and mice. Lhx4 and Prop1 mutants exhibit severe anterior pituitary hypoplasia resulting from limited differentiation and expansion of most specialized cell types. Little is known about the mechanism through which these genes promote pituitary development. In this study we determined that the hypoplasia in Lhx4 mutants results from increased cell death and that the reduced differentiation is attributable to a temporal shift in Lhx3 activation. In contrast, Prop1 mutants exhibit normal cell proliferation and cell survival but show evidence of defective dorsal-ventral patterning. Molecular genetic analyses reveal that Lhx4 and Prop1 have overlapping functions in early pituitary development. Double mutants exhibit delayed corticotrope specification and complete failure of all other anterior pituitary cell types to differentiate. Thus, Lhx4 and Prop1 have critical, but mechanistically different roles in specification and expansion of specialized anterior pituitary cells.

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CREB trans-activates the murine H+-K+-ATPase α2-subunit gene

AJP Cell Physiology ◽

10.1152/ajpcell.00065.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. C903-C911 ◽

Cited By ~ 10

Author(s):

Xiangyang Xu ◽

Wenzheng Zhang ◽

Bruce C. Kone

Keyword(s):

Dna Binding ◽

Transcriptional Control ◽

Luciferase Activity ◽

Collecting Duct ◽

Binding Activity ◽

Dominant Negative ◽

Proximal Promoter ◽

Mrna Levels ◽

Dna Binding Activity

Despite its key role in potassium homeostasis, transcriptional control of the H+-K+-ATPase α2-subunit (HKα2) gene in the collecting duct remains poorly characterized. cAMP increases H+-K+-ATPase activity in the collecting duct, but its role in activating HKα2 transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HKα2 promoter confers basal collecting duct-selective expression. This region contains several potential cAMP/Ca2+-responsive elements (CRE). Accordingly, we examined the participation of CRE-binding protein (CREB) in HKα2 transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HKα2 mRNA levels, and CREB overexpression stimulated the activity of HKα2 promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HKα2 promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HKα2 promoter-luciferase activity, suggesting that constitutive CREB participates in basal HKα2 transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HKα2 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. In vitro DNase I footprinting and gel shift/supershift analysis of the proximal promoter with recombinant glutathione S-transferase (GST)-CREB-1 and mIMCD-3 cell nuclear extracts revealed sequence-specific DNA-CREB-1 complexes at −86/−60. Mutation at three CRE-like sequences within this region abolished CREB-1 DNA-binding activity and abrogated CREB-VP16 trans-activation of the HKα2 promoter. In contrast, mutation of the neighboring −104/−94 κβ element did not alter CREB-VP16 trans-activation of the HKα2 promoter. Thus CREB-1, binding to one or more CRE-like elements in the −86/−60 region, trans-activates the HKα2 gene and may represent an important link between rapid and delayed effects of cAMP on HKα2 activity.

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A PIT-1 Homeodomain Mutant Blocks the Intranuclear Recruitment Of the CCAAT/Enhancer Binding Protein α Required for Prolactin Gene Transcription

Molecular Endocrinology ◽

10.1210/me.2001-0222 ◽

2003 ◽

Vol 17 (2) ◽

pp. 209-222 ◽

Cited By ~ 37

Author(s):

John F. Enwright ◽

Margaret A. Kawecki-Crook ◽

Ty C. Voss ◽

Fred Schaufele ◽

Richard N. Day

Keyword(s):

Gene Transcription ◽

Binding Protein ◽

Hormone Deficiency ◽

Centromeric Heterochromatin ◽

Pituitary Hormone ◽

Homeodomain Protein ◽

Pituitary Cells ◽

Enhancer Binding Protein ◽

Ccaat Enhancer Binding Protein ◽

Prl Gene

Abstract The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, including CCAAT/enhancer binding protein α (C/EBPα), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBPα with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBPα induced PRL gene transcription in pituitary GHFT1–5 cells, whereas the coexpression of Pit-1 and C/EBPα in HeLa cells demonstrated their cooperativity at the PRL promoter. Individually expressed Pit-1 or C/EBPα, fused to color variants of fluorescent proteins, occupied different subnuclear compartments in living pituitary cells. When coexpressed, Pit-1 recruited C/EBPα from regions of transcriptionally quiescent centromeric heterochromatin to the nuclear regions occupied by Pit-1. The homeodomain region of Pit-1 was necessary for the recruitment of C/EBPα. A point mutation in the Pit-1 homeodomain associated with the syndrome of combined pituitary hormone deficiency in humans also failed to recruit C/EBPα. This Pit-1 mutant functioned as a dominant inhibitor of PRL gene transcription and, instead of recruiting C/EBPα, was itself recruited by C/EBPα to centromeric heterochromatin. Together our results suggest that the intranuclear positioning of these factors determines whether they activate or silence PRL promoter activity.

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The CUP2 gene product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.4091-4095.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 4091-4095 ◽

Cited By ~ 1

Author(s):

C Buchman ◽

P Skroch ◽

J Welch ◽

S Fogel ◽

M Karin

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Regulatory Gene ◽

Point Mutations ◽

Dna Binding Protein ◽

Binding Activity ◽

Dna Binding Domain ◽

Yeast Saccharomyces Cerevisiae ◽

Dna Binding Activity ◽

Yeast Strains

CUP2 is a regulatory gene controlling expression of CUP1, which encodes the Cu-binding yeast metallothionein. CUP2, which is identical to the ACE1 gene, encodes a Cu-regulated DNA-binding protein. The CUP2 protein contains a cysteine-rich DNA-binding domain dependent on Cu+ and Ag+ ions which bind the cysteine residues and direct the refolding of the metal-free apoprotein. CUP2 mutant alleles from Cu-sensitive yeast strains have point mutations affecting the DNA-binding activity. These results establish CUP2 as the primary sensor of intracellular Cu+ in the yeast Saccharomyces cerevisiae, functioning as a Cu+-regulated transcriptional activator.

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Transcriptional Control of the Mycobacterial embCAB Operon by PknH through a Regulatory Protein, EmbR, In Vivo

Journal of Bacteriology ◽

10.1128/jb.188.8.2936-2944.2006 ◽

2006 ◽

Vol 188 (8) ◽

pp. 2936-2944 ◽

Cited By ~ 69

Author(s):

Kirti Sharma ◽

Meetu Gupta ◽

Monika Pathak ◽

Nidhi Gupta ◽

Anil Koul ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Control ◽

Regulatory Protein ◽

Binding Activity ◽

In Vivo Studies ◽

Promoter Regions ◽

Signaling Mechanism ◽

Dna Binding Activity

ABSTRACT EmbR, a putative transcriptional regulator from Mycobacterium tuberculosis, is homologous to the OmpR class of transcriptional regulators that possess winged helix-turn-helix DNA binding motifs. In contrast to other OmpR-like response regulators that are usually phosphorylated and controlled by histidine kinases, EmbR was recently shown to be phosphorylated by the cognate mycobacterial serine/threonine kinase PknH. Despite the in vitro evidence of phosphorylation and interaction between the kinase and regulator, the physiological function of the PknH-EmbR pair is still unknown. We identify the embCAB operon encoding arabinosyltransferases in M. tuberculosis as the cellular target of EmbR. Phosphorylation of EmbR enhances its DNA binding activity towards promoter regions of embCAB genes. In vivo studies involving expression of PknH in Mycobacterium smegmatis established its positive regulatory effect on transcription of the embCAB operon via phosphorylation of EmbR. Interestingly, increased transcription of embC, catalyzing arabinosylation of lipomannan (LM) to lipoarabinomannan (LAM), results in a high LAM/LM ratio, which in turn is a crucial factor in mycobacterial virulence. The PknH-mediated increase in the transcription of embAB genes significantly alters resistance to ethambutol, a frontline antituberculosis drug known to target embAB genes. These findings and in vivo upregulation of PknH inside the host macrophages suggest a functionally relevant signaling mechanism involving the PknH-EmbR-embCAB system.

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PROP1 Gene Screening in Patients with Multiple Pituitary Hormone Deficiency Reveals Two Sites of Hypermutability and a High Incidence of Corticotroph Deficiency

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.86.9.7811 ◽

2001 ◽

Vol 86 (9) ◽

pp. 4529-4535 ◽

Cited By ~ 51

Author(s):

S. Vallette-Kasic ◽

A. Barlier ◽

C. Teinturier ◽

A. Diaz ◽

M. Manavela ◽

...

Keyword(s):

Point Mutations ◽

Hormone Deficiency ◽

Heterozygous Mutation ◽

Pituitary Hormone ◽

Gene Encoding ◽

Exon 2 ◽

High Incidence ◽

Prop1 Gene ◽

Gene Alterations

Alterations of the gene encoding the pituitary transcription factor PROP1 were associated with congenital forms of multiple pituitary hormone deficiencies in several families. Among 23 patients with multiple pituitary hormone deficiencies screened for a PROP1 gene abnormality, nine belonging to eight unrelated families had homozygous PROP1 gene defects. All mutations were located in exon 2 and affected only two different sites: a homozygous AG deletion at codons 99/100/101 (n = 5); homozygous point mutations affecting codon 73: R73C (n = 2) or R73H (n = 1), and a R73C/R99X double-heterozygous mutation (n= 1). R73H and R99X were never described. All patients were born to unaffected parents, and consanguinity was documented in two patients. They had complete GH, LH-FSH, and TSH deficiencies and normal basal levels of PRL. Delayed ACTH deficiency was diagnosed in four of nine patients. At magnetic resonance imaging the anterior pituitary was hypoplastic in seven patients and hyperplastic in two. This study found two novel mutations (R73H and R99X) and underlines the high incidence of PROP1 gene alterations in patients with multiple pituitary hormone deficiencies. A corticotroph deficiency was frequently observed in association with GH, TSH, and gonadotropin deficiencies and should be carefully sought during follow-up.

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Lhx4 Deficiency: Increased Cyclin-Dependent Kinase Inhibitor Expression and Pituitary Hypoplasia

Molecular Endocrinology ◽

10.1210/me.2014-1380 ◽

2015 ◽

Vol 29 (4) ◽

pp. 597-612 ◽

Cited By ~ 6

Author(s):

Peter Gergics ◽

Michelle L. Brinkmeier ◽

Sally A. Camper

Keyword(s):

Kinase Inhibitor ◽

Developmental Regulation ◽

Hormone Deficiency ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Cyclin Dependent Kinase ◽

Cyclin Dependent Kinase Inhibitor ◽

Show Evidence ◽

Progenitor Cell Proliferation ◽

Pituitary Hypoplasia

Abstract Defects in the Lhx4, Lhx3, and Pitx2 genes can cause combined pituitary hormone deficiency and pituitary hypoplasia in both humans and mice. Not much is known about the mechanism underlying hypoplasia in these mutants beyond generally increased cell death and poorly maintained proliferation. We identified both common and unique abnormalities in developmental regulation of key cell cycle regulator gene expression in each of these three mutants. All three mutants exhibit reduced expression of the proliferative marker Ki67 and the transitional marker p57. We discovered that expression of the cyclin-dependent kinase inhibitor 1a (Cdkn1a or p21) is expanded dorsally in the pituitary primordium of both Lhx3 and Lhx4 mutants. Uniquely, Lhx4 mutants exhibit reduced cyclin D1 expression and have auxiliary pouch-like structures. We show evidence for indirect and direct effects of LHX4 on p21 expression in αT3-1 pituitary cells. In summary, Lhx4 is necessary for efficient pituitary progenitor cell proliferation and restriction of p21 expression.

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Effects of Denys−Drash Syndrome Point Mutations on the DNA Binding Activity of the Wilms' Tumor Suppressor Protein WT1†

Biochemistry ◽

10.1021/bi960758o ◽

1996 ◽

Vol 35 (37) ◽

pp. 12070-12076 ◽

Cited By ~ 37

Author(s):

Franck Borel ◽

Kathleen C. Barilla ◽

Tatyana B. Hamilton ◽

May Iskandar ◽

Paul J. Romaniuk

Keyword(s):

Tumor Suppressor ◽

Dna Binding ◽

Wilms Tumor ◽

Point Mutations ◽

Binding Activity ◽

Tumor Suppressor Protein ◽

Dna Binding Activity ◽

Drash Syndrome

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Inhibition of Interferon Regulatory Factor 7 (IRF7)-Mediated Interferon Signal Transduction by the Kaposi's Sarcoma-Associated Herpesvirus Viral IRF Homolog vIRF3

Journal of Virology ◽

10.1128/jvi.00235-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8282-8292 ◽

Cited By ~ 91

Author(s):

Chul Hyun Joo ◽

Young C. Shin ◽

Michaela Gack ◽

Liguo Wu ◽

David Levy ◽

...

Keyword(s):

Dna Binding ◽

Antiviral Activity ◽

Viral Infection ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Binding Activity ◽

Dna Binding Activity ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Α Helix ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-α) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double α helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double α helix motif-containing peptide effectively suppresses IRF7-mediated IFN-α production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.

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