A 60-bp Core Promoter Sequence of Murine Lactate Dehydrogenase C is Sufficient to Direct Testis-Specific Transcription in Vitro1

Abstract

A clone containing the 5' flanking region of the testis-specific murine lactate dehydrogenase C (Ldhc) gene was isolated from a mouse genomic library. Promoter activity was demonstrated within a 720-bp fragment in testis nuclear extract (TN). Interestingly, the addition of liver nuclear extract (LN) significantly repressed Ldhc promoter activity in the transcription assay system. Sequence analysis of this promoter region revealed several ubiquitous cis-regulatory elements, including one TATA box, one GC box, and two putative CCAAT elements. Analysis of a series of deletion mutants revealed that a 60-bp core promoter sequence was sufficient to direct basal, testis-specific transcription in an in vitro transcription system. A 103-kDa protein in TN and a 65-kDa protein in LN bind to the same palindromic sequences within the 60-bp core promoter region.

Transgenic Mice Demonstrate a Testis-specific Promoter for Lactate Dehydrogenase, LDHC

“…Data have been obtained to support the regulatory role of a palindromic sequence overlapping the TATA box and extending 3Ј to the transcription initiation site. A 103-kDa protein in testis nuclear extract and a 65-kDa protein in liver nuclear extract binds to this palindromic sequence within the 60-bp core promoter region (3). From mutational analyses, we have established the palindrome as a dual function regulatory region necessary for promoter activity in testis nuclear extract and as responsible for repression of the promoter in liver nuclear extract (4).…”

“…A 720-bp mouse ldhc fragment (Ϫ710 to ϩ12) for construct 100H␤ was amplified by PCR as described previously (3). Similarly, the 700-bp fragment for construct 100⌬H␤ was amplified as described in Zhou and Goldberg (4).…”

ldhc Expression in Non-germ Cell Nuclei Is Repressed by NF-I Binding

“…The livers were dissected from 15 mice, minced, and homogenized in buffer as described previously (2). Testes from 50 mice were decapsulated, minced, and homogenized.…”

“…1A shows the sequence of the 430-bp mouse ldhc promoter and the location of the palindrome (inverted arrows), which overlaps the TATA box and includes the transcription initiation site (indicated by an asterisk). Previous studies revealed that the palindrome bound a 65-kDa protein from liver nuclear extracts (2). In vitro transcription assays demonstrated that mutations in the 5Ј region of the palindrome resulted in low activity with liver nuclear extract in what otherwise would be a silent mldhc promoter (4).…”

“…The palindrome sequence binds proteins from testis and liver nuclear extracts in electrophoretic mobility shift assays (EMSA). Southwestern analysis reveals binding to a 105-kDa protein extracted from testis nuclei and a 65-kDa protein from liver nuclear extracts (2). Functional activity has been demonstrated by in vitro transcription assays using a promoter fragment with either the wild-type sequence or with random mutations introduced in the center or in the 5Ј-flanking region of the palindrome (4).…”