HIGH-MOBILITY GROUP PROTEIN-14 AND PROTEIN-17 CAN SPACE NUCLEOSOMAL PARTICLES DEFICIENT IN HISTONES H2A AND H2B CREATING A TEMPLATE THAT ISTRANSCRIPTIONALLY ACTIVE

Recently, using a well defined nucleosomal assembly system, we demonst rated that high mobility group proteins (HMGs) 14 and 17 can organize nucleosomes into a regular array with a nucleosomal repeat length of 1 60-165 base pairs in vitro. Interestingly, such a short repeat length has been described for lower eukaryotes and for active chromatin. To b egin to investigate how these proteins may prevent the close packing o f nucleosomes, assembly reactions were carried out in which the relati ve amounts of HMGs 14 and 17, histones H2A and H2B, and the N1/N2-(H3, H4) complex were varied in assembly reactions. Under conditions in wh ich histones H2A and H2B were Limiting and in the absence of HMGs 14 a nd 17, micrococcal nuclease digestion of the assembled product produce d a ladder of DNA fragments that was much less well defined and which included DNA that was associated with subnucleosomal structures. The a pparent repeat length for this chromatin template was around 125 base pairs. Most interestingly, when HMGs 14 and 17 were added to this asse mbly reaction, ''nucleosome-like'' structures were reassembled as show n by the restoration of a regular, well defined ladder of DNA fragment s upon micrococcal nuclease digestion. The apparent repeat length incr eased from 125 to approximately 145 base pairs. Analysis of the protei n composition of chromatin formed in the presence or absence of HMGs 1 4 and 17 reveals that HMGs 14 and 17 might be able to substitute for a histone H2A-H2B dimer in a H2A/H2B-deficient nucleosome. The ability to form a regularly spaced nucleosomal template is also lost when exce ss HMGs 14 and 17 are used in assembly reactions. Spacing can be resto red by the addition of poly(glutamate,alanine), a chemical polymer of negative charge, which may indicate that carrier proteins (specific or nonspecific) may be required for the proper incorporation of all chro matin assembly components into chromatin in vivo. Finally, although th e mechanism of action is not known, HMGs 14 and 17 can partially overc ome inhibition of initiation of transcription caused by the formation of nucleosomal particles deficient in histones H2A and H2B.