Farhad Marashi

Human histone genes are interspersed with members of the Alu family and with other transcribed sequences.

Abstract We have isolated a series of recombinant λCh4A phages containing human histone genes. Histone H2A, H2B, H3 and H4 genes have been found to be clustered, but are not present in any simple repeat pattern. Hybridization of a blot containing phage DNA with S phase polysomal cDNA indicates the presence of additional sequences complementary to HeLa polysomal RNA sequences. Northern blot analysis using these clones as probes has also shown the presence of sequences complementary to non-histone-coding RNAs, some of which accumulate differentially in different stages of the cell cycle. We have also found, by hybridization with appropriate probes, that histone genes are interspersed with several copies of the Alu DNA family; however, not all of the histone genes are associated with an Alu DNA sequence.

Organization and cell cycle regulation of human histone genes

Histone genes represent a moderately repeated set of genes in human cells. To study the organization and regulation of human histone genes, we have characterized a series of recombinant lambda Charon 4A phage containing genomic human histone sequences (designated XHHG) . Our analyses indicate that human histone genes are clustered, but are not organized in a simple tandem repeat pattern, as is observed for several lower eukaryotes. For example, several of the human genomic fragments we have isolated contain two each of segments coding for H3 and H4 histones. Of particular interest with respect to organization and expression of the human histone genes is the presence of at least sevem different H4 histone mRNAs associated with polysomes of S-phase HeLa cells. At least three of the HeLa H4 histone mRNAs are products of distinct genes and two H4 histone genes in the same genomic fragment code for different H4 mRNAs. We have used our cloned human histone genes to examine the regulation of histone gene expression in human cells. Although it is generally agreed that histone protein synthesis in HeLa cells is restricted to the S-phase of the cell cycle, and therefore parallels DNA replication, both transcriptional and posttranscriptional levels of control have been postulated. By probing electrophoretically fractionated, filter-immobilized RNAs with several cloned genomic human histone sequences representing different histone gene clusters, we have assessed the steady-state levels of histone RNAs in the nucleus and cytoplasm of GIand S-phase HeLa S3 cells. The representation of histone mRNA sequences in G, compared with S-phase cells was less than 1% in the cytoplasm and approximately 1% in the nucleus. These data are consistent with control occurring primarily at the transcriptional level, but we cannot dismiss the possibility that regulation of histone gene expression is to some extent, and/or under some biological circumstances, mediated post-transcriptionally. If histone gene transcription does occurs in G,, the RNAs must either be rapidly degraded or be transcribed to a limited extent compared with those of the S phase. An unexpected result was obtained when a northern gel blot of cytoplasmic RNA from GIand S-phase cells was hybridized with XHHG41 DNA (containing H3 and H4 genomic human histone sequences). This clone hybridizes with histone mRNAs present in S-phase cytoplasmic RNA, but also hybridizes with a G, cytoplasmic RNA approximately 330 nucleotides in length. This RNA,

Regulation of specific genes during the cell cycle. Utilization of homologous cDNAs and cloned sequences for studying histone gene expression in human cells

Evidence for differential gene expresion during the cell cycle and approaches for studying cell-cycle-stage specific gene expression are summarized. Attention is focused on regulation of histone gene expression during the cell cycle of continuously dividing cells and after stimulation of nondividing cells to proliferate. The level(s) at which control of histone gene expression occurs and the possible involvement of chromosomal proteins in the regulation of histone gene expression are discussed. The preparation of cloned human histone sequences and their use in studying the structural and functional properties of human histone genes are presented.

Organization and Cell Cycle Periodic Expression of Human Histone Genes

In this chapter we will summarize several of the experimental approaches we have been taking to examine human histone genes. The structure and organization of human histone genes will be discussed, particularly within the context of the putative relationships of specific regions of the genes to their expression. Approaches to assessing the levels at which control of histone gene expression resides will also be considered. Results will be presented which suggest that: a) Human histone genes are a family of moderately reiterated sequences with variations in the structure, organization, and possibly in the regulation of the various copies. b) At least 15 different, though not necessarily all, human histone genes are coordinately expressed during the S phase of the cell cycle and appear to be temporally and functionally coupled with DNA replication. c) There are both transcriptional and post-transcriptional components to the regulation of those histone genes expressed in conjunction with DNA replication.

Selective expression of histone genes in mouse-human hybrid cells.

Mouse-human hybrid cells preferentially segregating mouse chromosomes contain predominantly human histone mRNAs and synthesize human histone proteins. In contrast, hybrids segregating human chromosomes contain both human and murine histone mRNAs, yet synthesize only mouse histone proteins. These results suggest transcriptional control of histone gene expression in hybrids segregating mouse chromosomes and post-transcriptional regulation in hybrids segregating human chromosomes.