SIN1 interacts with a protein that binds the URS1 region of the yeastHOgene

Abstract

Evidence has recently been mounting suggesting that a number of chromatin components previously thought to primarily or exclusively have structural function, also have a regulatory role in eukaryotic transcription. Notably, in yeast, histone H4 N-terminal sequence has been shown to be required for promoter activation of certain genes in vivo, and mutations in histone H3 (SIN2) or in SIN1 (which has some sequence similarity to HMG1) are able to suppress swi1, swi2, and swi3 mutations, restoring transcription to HO as well as a number of other genes. In this paper we report the identification of a novel protein or protein complex that specifically binds a short sequence in the HO regulatory region on the one hand, and on the other somehow appears to contact the SIN1 protein. We have shown that the DNA binding activity itself does not contain SIN1, since extracts from sin1 delta strains retain the activity. Interestingly, extracts made from cells carrying the dominant sin1-2 point mutation lack the binding activity. Furthermore, bacterially produced sin1-2 protein can dissociate a DNA/protein complex while a similarly produced SIN1 protein has no effect on the complex at similar concentrations. When the DNA sequence to which the protein complex binds is placed in a CYC1 promoter lacking a UAS (upstream activating sequence), it can serve as a weak UAS in a SIN1 dependent way. Our data imply that a sequence specific DNA binding protein(s) may mediate between the SIN1 protein and the basal transcription apparatus transcribing HO.

FER Kinase Activation of Stat3 Is Determined by the N-terminal Sequence

“…Electromobility Gel Shift Assay-A double-stranded synthetic fragment that carries the Stat3 binding site (29) was labeled with [␣-32 P]␣ATP by fill in reaction using Klenow fragment and was then purified on 12% acrylamide gel (30). 10-g nuclear protein extracts were incubated at 25°C for 15 min with 400 ng of poly(dI⅐dC) and 0.1 ng of labeled probe in the following binding buffer: 4 mM Tris-HCl, pH 8.0, 40 mM NaCl, 4 mM MgCl 2 , and 5% glycerol in total volume of 25 l. The assay mixture was then separated on 4% acrylamide Tris-borate EDTA gel at 100 V for 2-3 h. The gel was dried and exposed overnight on Kodak film (X-OMAT AR).…”

Functional Domains of the Yeast Chromatin Protein Sin1p/Spt2p Can Bind Four-way Junction and Crossing DNA Structures

“…The C-terminal Domain GST-Sin1p-(303-333) Is Also Able to Bind 4WJDNA-The C-terminal domain, amino acids 303-333, is known to contain functional activity since a change in amino acid 314 from glutamic acid to lysine eliminates sin1 suppression of swi1 mutants (4,9). Furthermore a series of mutations in this domain caused phenotypic changes in the SPT phenotype (8).…”

“…It is estimated that there are about 1200 (7) to 2000 (8) copies of the protein molecule per cell, indicating that it probably binds the chromatin at multiple locations. It was shown that in vitro the N terminus of Sin1p/Spt2p protein interacts with the tetratricopeptide repeat domains of Cdc23p, a protein involved in chromosome segregation, whereas the C terminus of Sin1p/Spt2p binds proteins involved in transcriptional regulation (9,10).…”

Characterization of a short unique sequence in the yeast HO gene promoter that regulates HO transcription in a SIN1 dependent manner

“…The gel retardation assay was performed as described [4] except that competing unlabeled DNA was added as described in the legend to …”

“…In sin1-2 mutants, HO is transcribed regardless of the presence of the $WI1, SW12, SWI3 positive regulators [3]. Interestingly, sinl-2 mutant protein is able to specifically remove the protein that binds the XBS in vitro [4]. SIN 1 has been shown to be a DNA *Corresponding author.…”