Msx1 homeodomain transcription factor and TATA-binding protein interact to repress the expression of the glycoprotein hormone α subunit gene

Although there is evidence that the LIM homeodomain transcription factor, Lhx2, can stimulate transcription of the glycoprotein hormone α-subunit gene, the role of Lhx2 in regulating TSH β-subunit has not been established. In the present studies, the ability of Lhx2 to regulate transcription of the TSH β-subunit gene was examined. In the thyrotrope-derived TαT1 cell line, Lhx2 expression was found to be induced by treatment with either TRH or cAMP, consistent with the possibility that Lhx2 may play a role in mediating the ability of this signaling pathway to stimulate TSH gene expression. Transient, forced overexpression of Lhx2 stimulated activity of a TSH β-subunit reporter gene. Deletion studies provided evidence that the −177 to −79 region of the TSH β-subunit promoter was necessary for stimulation of reporter gene activity by Lhx2. A gel mobility shift assay provided the evidence that Lhx2 can bind to this region of DNA. DNase I footprinting studies demonstrated that two distinct regions of the TSHβ promoter, −118 to −108 and −86 to −68, are protected by Lhx2 from nuclease digestion. These regions contain repeats of the sequence, 5′-(G/T)CAAT(T/A)-3′. Mutation of this sequence, especially in the −86 to −68 region, substantially decreased Lhx2 responsiveness of the TSH β-subunit reporter gene. In addition, a DNA fragment containing the −177 to −79 region of the TSHβ promoter was found to confer Lhx2 responsiveness to a minimal promoter. These results provide multiple lines of evidence consistent with a role for Lhx2 in modulating expression of the TSH β-subunit gene.

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Multiple Characteristics of a Pentameric Regulatory Array Endow the Human α-Subunit Glycoprotein Hormone Promoter with Trophoblast Specificity and Maximal Activity

Molecular Endocrinology ◽

10.1210/mend.11.11.0007 ◽

1997 ◽

Vol 11 (11) ◽

pp. 1669-1680

Author(s):

Paul R. Budworth ◽

Patrick G. Quinn ◽

John H. Nilson

Keyword(s):

Binding Protein ◽

Maximal Activity ◽

Regulatory Elements ◽

Subunit Gene ◽

Camp Response Element ◽

Glycoprotein Hormone ◽

Specific Expression ◽

Α Subunit ◽

Multiple Characteristics ◽

Ccaat Box

Abstract Trophoblast-specific expression of the humanα -subunit glycoprotein hormone gene requires a tightly linked array of five different regulatory elements [trophoblast-specific element (TSE), α-activating element (αACT), a tandem cAMP response element (CRE), junctional regulatory element (JRE), and a CCAAT box]. We examined their contextual contributions to trophoblast-specific expression by using transfection assays to evaluate activity of systematic block replacement mutations made within the 1500-bp 5′-flanking region of the human α-subunit gene. While all five elements were required for full activity, only the TSE and JRE displayed trophoblast specificity. Interestingly, the TSE-binding protein has limited tissue distribution whereas a JRE-binding protein appears trophoblast specific. Likewise, replacement studies with an AP-1 element that binds heterodimers of jun and fos indicated that this element was incapable of compensating for either the tandem CRE or JRE. This preference for both CRE- and JRE-binding proteins provides another avenue for configuring an α-subunit promoter with trophoblast specificity. Additional analysis with a cAMP response element binding protein (CREB)-Gal4 fusion protein further underscored the importance of CREB as well as suggested that transcriptional contributions come from both the DNA-binding domain and transactivation domain of this protein. We also examined the interactive nature of the pentameric array by placing a 15-bp random sequence between each element. Remarkably, only the insertion 3′ of the CCAAT box diminished promoter activity. This suggested the absence of direct interactions between the transcriptional factors that bind each element in the array. It also suggested that the CCAAT box is position-dependent relative to the TATA box. This position dependence appeared cell-specific, as it was not manifest in a gonadotrope cell line (αT3–1 cells). Thus, the CCAAT box also has tissue-specific characteristics that assist in targeting expression of the α-subunit gene to trophoblasts. Together, these data suggest that multiple characteristics of a complex pentameric array of regulatory elements endow the α-subunit promoter with trophoblast specificity and maximal activity.

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