The cell cycle (left) is regulated by a variety of genetic and biochemical factors, many of which remain poorly understood.  One of the most intensive investigative efforts of cell and molecular biology is devoted to discovering the fundamental molecular switches that convert cells from cytoplasmic growth to cell division.  The motivation for this research is more than academic, because faulty cell cycle regulation leads to the uncontrolled growth characteristic of cancer.
        Recent research has begun to reveal the genes and gene products active and modified in cell cycle [review cell cycle] regulation.  The genes identified by this research fall roughly into three categories 1) Genes and gene products acting as fundamental controls that directly regulate passage through the cell cycle.  Particularly important among these genes are those controlling passage though the G1-S boundary and the transition from G2 to mitosis.  2) Genes encoding proteins that indirectly regulate passage through the cell cycle, for example cell surface receptors and growth factors.  3)  Genes that do not directly or indirectly regulate the cell cycle, but encode proteins and other factors required to maintain activities critical to the cell cycle, such as DNA replication.  Although genes in this third category  do not act as regulatory elements,  mutations or alterations of these genes can alter or stop the cell cycle.  (the above two paragraphs, including the figure at left, were paraphrased or directly copied from Introduction to Cell and Molecular Biology by Stephen Wolfe)
        The Marzluff Lab has two main areas of interest.  The first is histone genes, which are critical to DNA replication and thus fall into the third category above.  We are specifically interested in the post-transcriptional regulation of histone gene expression during the cell cycle.  The second area of interest is the G1 cyclin proteins (cyclins D and E) and their associated cyclin-dependent kinases, which fall into the first category above as proteins that directly regulate transitions between cell cycle phases.
 

Histone Genes and SLBP:
        In eukaryotic cells, the DNA double helix wraps around histone octamers to form the nucleosome, the basic structural unit of chromosome [review histones].  Each histone octamer contains two copies of each core histone, H2a, H2b, H3, and H4.  Histone H1 binds to the linker region between two nucleosomes helps the chromosome fold into a higher order structure. Histone proteins not only act as structural proteins, but modifications including phosphorylation, methylation and especially acetylation and deacetylation, also play very important roles in the regulation of gene expression and chromatin structure.
        During each cell cycle, a cell replicates its DNA once and only once in S phase.  At this time, cells must synthesize a complete set of histone proteins to package the newly replicated DNA.  In order to accomplish this, and also to coordinate the synthesis of different histone proteins, histone genes have some unique features.  Unlike all other metazoan (animal) mRNA's, mRNA's transcribed from replication-dependent histone genes do not contain introns and do not end in a 3' poly A tail.  Instead of a poly A tail, replication-dependent histone mRNA's end in a conserved 26-nucleotide sequence that contains a 16-nucleotide stem-loop (figure 1, right).  Processing of the 3' end of histone pre-mRNA occurs by endonucleolytic cleavage of the pre-mRNA on the 3' side of the stem-loop, releasing the mature mRNA from the chromatin template.  This cleavage requires several trans-acting factors (trans-acting = encoded on a different strand of DNA than the gene it is acting upon), including a small nuclear RNP (ribonucleic protein) called the U7 snRNP and a protein called the stem loop binding protein (SLBP), which binds the 26 nucleotide stem-loop sequence.   There are also probably additional, unknown factors required for cleavage.   One of the major regulatory events in the cell cycle is regulation of histone pre-mRNA processing, which is at least partially mediated by cell-cycle regulation of the levels of the SLBP protein.
        As cells enter S phase, the steady-state level of histone mRNA increases 30-50 fold. The transcription rate increases about 3-5 fold. The processing efficiency increases about 10 fold. At the end of S phase, the level of histone mRNAs quickly decreases due to the change of mRNA half-life [Figure 2:  graph of histone mRNA levels during cell cycle]. The unique stem-loop structure present at the end of all replication-dependent histone mRNA is essential for the pre-mRNA processing, nuclear export, histone mRNA translation, and degradation regulation. It also provides a way to the coordinate regulation for the expression of histone genes.
        Using the novel yeast three-hybrid screen method, Z.F.Wang in our lab cloned the stem-loop binding protein. He cloned SLBP cDNAs from mouse, human, and two cDNAs (XSLBP1, XSLBP2) from Xenopus.   We have also cloned one Drosophila SLBP cDNA and one apparent sea urchin SLBP. All these SLBP genes have a conserved 73 amino acid RNA binding domain located in the middle of the protein. This RNA binding domain has no similarity to any other known RNA binding sequences. Like the stem-loop structure, the SLBP is probably involved in every step of histone mRNA metabolism [Figure 3:  The Role of SLBP in Histone mRNA Metabolism].

The Cyclins:
        The cyclins and their associated cyclin dependent kinases (CDK's) are a group of proteins that 'work in shifts' to trigger successive stages of the cell cycle (left).  Sea urchins provide a convenient model system in which to examine these proteins.  Female sea urchins store their gametes as haploid eggs, which are naturally arrested in a "pseudo-G0"  state and proceed directly into S phase shortly after fertilization.  After fertilization, the embryos undergo six to seven cell divisions without gap phases.
        The reason sea urchins are convenient to study is that a large number of synchronously dividing embryos can be obtained by simply combining sea urchin eggs and sperm in a flask of sea water.  After fertilization, any number of experiments can be performed to study the activity of cyclins and their associated CDK's directly after fertilization (a G0 - S transition) as well as during subsequent embryo divisions.