PMID- 10451362
OWN - NLM
STAT- MEDLINE
DCOM- 19990922
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 38
IP  - 33
DP  - 1999 Aug 17
TI  - The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates
      the rat osteocalcin promoter.
PG  - 10678-90
AB  - Msx2 is a homeodomain transcriptional repressor that exerts tissue-specific
      actions during craniofacial skeletal and neural development. To identify
      coregulatory molecules that participate in transcriptional repression by Msx2, we
      applied a Farwestern expression cloning strategy to identify transcripts encoding
      proteins that bind Msx2. A lambdagt11 expression library from mouse brain was
      screened with radiolabeled GST-Msx2 fusion protein encompassing the core
      suppressor domain of Msx2. A cDNA was isolated that encodes a novel protein
      fragment that binds radiolabeled Msx2. Homeoprotein binding activity was
      confirmed by Farwestern analysis of the T7-epitope-tagged recombinant protein
      fragment, and interactions in vitro require Msx2 residues necessary for
      transcriptional suppression in vivo. On the basis of biochemical analyses, this
      novel protein was named MINT, an acronym for Msx2-interacting nuclear target
      protein. The original clone is part of a 12.6 kb transcript expressed at high
      levels in testis and at lower levels in calvarial osteoblasts and brain. Multiple
      clones isolated from a mouse testis library were sequenced to construct a MINT
      cDNA contig of 11 kb. Starting from an initiator Met in good Kozak context, a
      large nascent polypeptide of 3576 amino acids is predicted, in contiguous open
      reading frame with the Msx2 interaction domain residues 2070-2394. Protein
      sequence analysis reveals that MINT has three N-terminal RNA recognition motifs
      (RRMs) and four nuclear localization signals. Western blot analysis of
      fractionated cell extracts reveals that mature approximately 110 kDa (N-terminal)
      and approximately 250 kDa (C-terminal) MINT protein fragments accumulate in
      chromatin and nuclear matrix fractions, cosegregating with Msx2 and topoisomerase
      II. In gel shift assays, the MINT RRM domain selectively binds T- and G-rich DNA 
      sequences; this includes a large G/T-rich inverted repeat element present in the 
      proximal rat osteocalcin (OC) promoter, overlapping three cognates that support
      OC expression in osteoblasts. MINT and OC mRNAs are reciprocally regulated during
      differentiation of MC3T3E1 calvarial osteoblasts. Consistent with its proposed
      role as a nuclear transcriptional factor, transient expression of MINT(1-812)
      suppresses the FGF/forskolin-activated OC promoter, does not significantly
      regulate CMV promoter activity, but markedly upregulates the HSV thymidine kinase
      promoter in MC3T3E1 cells. In toto, these data indicate that the novel nuclear
      protein MINT binds the homeoprotein Msx2 and coregulates OC during craniofacial
      development. Msx2 and MINT both target an information-dense, osteoblast-specific 
      regulatory region of the OC proximal promoter, nucleotides -141 to -111. The
      N-terminal MINT RRM domain represents an authentic dsDNA binding module for this 
      novel vertebrate nuclear matrix protein. Acting as a scaffold protein, MINT
      potentially exerts both positive and negative regulatory actions by organizing
      transcriptional complexes in the nuclear matrix.
FAU - Newberry, E P
AU  - Newberry EP
AD  - Department of Medicine, Center for Cardiovascular Research, Division of
      Endocrinology, Diabetes, and Metabolism, Washington University School of
      Medicine, St. Louis, Missouri 63110, USA.
FAU - Latifi, T
AU  - Latifi T
FAU - Towler, D A
AU  - Towler DA
LA  - eng
SI  - GENBANK/AF156529
GR  - AR43731/AR/NIAMS NIH HHS/United States
GR  - DK52446/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (DNA, Complementary)
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (Homeodomain Proteins)
RN  - 0 (MSX2 protein)
RN  - 0 (Nuclear Proteins)
RN  - 0 (Peptide Fragments)
RN  - 0 (RNA-Binding Proteins)
RN  - 0 (Repressor Proteins)
RN  - 0 (Spen protein, mouse)
RN  - 0 (Trans-Activators)
RN  - 104982-03-8 (Osteocalcin)
RN  - 452VLY9402 (Serine)
RN  - 5Z93L87A1R (Guanine)
RN  - 9DLQ4CIU6V (Proline)
RN  - QR26YLT7LT (Thymine)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Cell Line
MH  - Cloning, Molecular
MH  - DNA, Complementary/isolation & purification
MH  - DNA-Binding Proteins/*metabolism
MH  - Guanine/metabolism
MH  - Homeodomain Proteins/*metabolism
MH  - Mice
MH  - Molecular Sequence Data
MH  - Molecular Weight
MH  - Nuclear Proteins/genetics/*metabolism/physiology
MH  - Osteoblasts/metabolism
MH  - Osteocalcin/*genetics/*metabolism
MH  - Peptide Fragments/metabolism
MH  - Proline/metabolism
MH  - *Promoter Regions, Genetic
MH  - RNA-Binding Proteins/*metabolism
MH  - Rats
MH  - Repressor Proteins/physiology
MH  - Serine/metabolism
MH  - Thymine/metabolism
MH  - Trans-Activators/physiology
EDAT- 1999/08/18 00:00
MHDA- 1999/08/18 00:01
CRDT- 1999/08/18 00:00
PHST- 1999/08/18 00:00 [pubmed]
PHST- 1999/08/18 00:01 [medline]
PHST- 1999/08/18 00:00 [entrez]
AID - 10.1021/bi990967j [doi]
AID - bi990967j [pii]
PST - ppublish
SO  - Biochemistry. 1999 Aug 17;38(33):10678-90. doi: 10.1021/bi990967j.