Cloning and functional analysis of a novel family of nuclear matrix transcription factors (NP /NMP4) that regulate type I collagen expression.

摘要

Collagen type I expression in connective tissue is coupled to cell shape which may be mediated by the tissue matrix. The tissue matrix system is the dynamic linkage between the structural proteins of the nucleus, the cytoplasm, and the extra-acellular matrix, that links DNA to the cell periphery. We propose that this substructure culminates with nuclear matrix architectural transcription factors, proteins that transform changes in cell shape into alterations in promoter geometry mid ultimately in gene expression. Architectural transcription factors typically bind within the minor groove of AT-rich DNA and bend it, modifying interactions between other transcription proteins.;We have identified the nuclear matrix architectural transcription factors, NP/NMP4. These proteins bind to the minor groove of two poly (dT) regions between --3482/--3463 bp (Site A) and --1574/--1555 bp (Site B) on the rat type I collagen alpha 1(I) polypeptide chain (COL1A1) promoter. In this study, our objectives were (i) to determine whether NP/NMP4 can induce DNA bending and (ii) to clone and characterize the NP/NMP4 cDNA. Circular permutation analysis revealed that NP/NMP4 induced DNA bending at the COL1A1 binding sites. Using Site B as a probe to screen a UMR-106 rat osteosarcoma expression library, we have isolated five individual clones. The expressed cloned proteins exhibited sequence-specific binding to COL1A1 Site B, and this binding was abolished by the AT-rich minor groove binding drug, Distamycin A, in a concentration dependent manner. Rabbit antisera raised against our expressed proteins supershifted the NP/NMP4-COL1A1 EMSA bands derived from osteoblastic nuclear matrix extracts. Sequence analyse of these clones revealed that NP/NMP4 are members of a novel Cys2His2 zinc finger protein family. This kind of zinc finger motif is a multifunctional domain involved in such diverse functions as gene regulation and nuclear matrix interaction. NP/NMP4 proteins also have an AT-hook domain, common to the HMG-I(Y) architectural transcription factors.;The present data answered our questions concerning the expression of NP/NMP4; NP/NMP4 homologues were observed in multiple mammalian species and these RNAs were expressed in rat embryonic bone and nerve. Also, they were detected in numerous adult tissues. These studies provided insights into our questions concerning the functional significance of these proteins. The transient co-transfection of UMR-106 cells with NP/NMP4 expression vectors and a COL1A1 promoter-reporter construct resulted in a decrease in the reporter gene activity. Additionally, NP/NMP4 expression was regulated by parathyroid hormone in UMR-106 cells. We conclude that NP/NMP4 are novel nuclear matrix transcription factors that contribute to collagen expression in bone and may be part of a general mechanical pathway that couples cell structure and function.