ldhc expression in non-germ cell nuclei is repressed by NF-I binding

Area of Science:

• Molecular Biology
• Gene Regulation
• Biochemistry

Background:

• The mouse lactate dehydrogenase C (mldhc) gene exhibits testis-specific expression, necessitating germ cell activation and somatic cell repression.
• A 60-bp promoter fragment upstream of the transcription start site shows testis-restricted activity in vitro.
• This fragment contains a TATA box and an overlapping 31-bp palindromic sequence.

Purpose of the Study:

• To investigate the role of the palindromic sequence in repressing mldhc gene expression in somatic tissues.
• To identify proteins that bind to the palindromic sequence and elucidate their function in mldhc gene regulation.

Main Methods:

• Gel retardation assays to detect protein binding sites within the palindrome.
• Site-directed mutagenesis of the NF-I binding site within the promoter.
• Transient transfection assays in mouse L cells using wild-type and mutated promoter-reporter constructs.
• Overexpression of NF-I proteins (NF-IA, -B, -C, -X) in transfected cells.

Main Results:

• Gel retardation assays identified two protein binding sites within the palindrome, with one site binding an NF-I/CTF family member.
• Mutation of the NF-I binding site in the promoter fragment resulted in a 4-fold increase in activity in mouse L cells.
• Overexpression of NF-I proteins decreased wild-type promoter activity by 20-50% but had no effect on the mutated promoter.

Conclusions:

• The NF-I proteins play a significant role in the repression of the mouse lactate dehydrogenase C (mldhc) gene in somatic cells.
• The palindromic sequence, through NF-I binding, acts as a silencer element for the mldhc gene in non-germline tissues.