Deposition of chromosomal protein HMG-17 during replication affects the nucleosomal ladder and transcriptional potential of nascent chromatin.

Abstract

A cell‐free system from Xenopus eggs was used to study the role of chromosomal protein HMG‐17 in the generation of the chromatin structure of transcriptionally active genes. Addition of HMG‐17 protein to the extracts, which do not contain structural homologs of the HMG‐14/−17 protein family, indicates the protein is incorporated into the nascent template during replication, prior to completion of chromatin assembly. The protein binds to and stabilizes the structure of the nucleosomal core thereby improving the apparent periodicity of the nucleosomal spacing of nascent chromatin. Assembly of HMG‐17 into the nascent chromatin structure significantly increased the transcription potential of the 5S RNA gene and satellite I chromatin. Kinetic studies indicate that the increase in transcriptional potential is observed only when HMG‐17 is incorporated into nucleosomes during chromatin assembly.

HMG17 is a chromatin-specific transcriptional coactivator that increases the efficiency of transcription initiation.

“…2E). This experiment revealed that there was no apparent depletion of H2A-H2B upon incorporation of HMG17 into the chromatin, which is consistent with the results of previous studies in which the protein composition of HMG 17-containing chromatin was examined (see, e.g., Albright et al 1980;Shick et al 1985;Crippa et al 1993).…”

“…Hence, these results indicate that the stimulation of GAL4-VP 16-activated transcription by HMG 17 is chromatin-dependent, which suggests that the specific binding of HMG17 to nucleosomal DNA, as characterized previously ( Fig. 2D; Albright et al 1980;Mardian et al 1980;Sandeen et al 1980;Crippa et al 1992Crippa et al , 1993A1-HMG17 is a t r a n s c r i p t i o n a l c o a c t i v a t o Binding of GAL4{1-147)to the template is not sufficient for transcriptional potentiation by HMG17. GAL4(1-147)contains the amino-terminal 147 amino acid residues of the yeast GAL4 protein and is identical to GAL4-VP16, except that it does not contain the transcriptional activation domain derived from the herpesvims VP16 protein.…”

“…2D). In this assay, the specific binding of either HMG14 or HMG17 to core particles results in a reduction in the mobility of core particles during native gel electrophoresis (Albright et al 1980;Mardian et al 1980;Sandeen et al 1980;Crippa et al 1992Crippa et al , 1993Alfonso et al 1994). Chromatin was assembled in the presence or absence of HMG17, purified by sucrose gradient sedimentation, digested extensively (to core particles) with micrococcal nuclease, and then subjected to electrophoresis on a nondenaturing polyacrylamide gel.…”

“…HMG14 and HMG17 are small (---10 kD), highly charged proteins that interact with high affinity to two distinct sites in nucleosomal cores (Albright et al 1980;Mardian et al 1980;Sandeen et al 1980;Shick et al 1985;Cook et al 1986;Crippa et al 1992Crippa et al , 1993Alfonso et al 1994). Moreover, HMG14/17 proteins bind with higher affinity to core particles than to naked DNA (Sandeen et al 1980).…”

“…Biochemical experiments with SV40 minichromosomes had led to the conclusion that HMG14 stimulates transcriptional elongation but not initiation (Ding et al 1994). In addition, studies of HMG17-containing chromatin templates had indicated that HMG17 stimulates transcription by RNA polymerase III (Crippa et al 1993;Trieschmann et al 1995). Thus, the available data suggest a role for HMG14/17 in transcriptional regulation.…”