Jeffrey A. Kramer

Nuclear Matrix Interactions within the Sperm Genome

Analysis of the haploid-expressed human PRM1 PRM2 TNP2 genic domain has revealed two regions of attachment to the sperm nuclear matrix. These sperm nuclear matrix attachment regions delimit the DNase I-sensitive domain of this haploid-expressed locus. The domain is intermediately associated with but not attached to the nuclear matrix. DNase I-sensitive genes within the mature sperm nucleus, such as protamine 1, protamine 2, transition protein 2, α-globin, and β-actin, display this intermediate affinity for the sperm nuclear matrix. This may denote their role in templating the male genome prior to fertilization, thus ensuring the formation of a viable male pronucleus during early embryonic development.

Reprogramming the male gamete genome: a window to successful gene therapy

Hematopoiesis and spermatogenesis both initiate from a stem cell capable of renewal and differentiation. Each pathway reflects the expression of unique combinations of facultative, i.e. tissue-specific and constitutive, i.e. housekeeping, genes in each cell type. In spermatogenesis, as in hematopoiesis, commitment is mediated by the mechanism of potentiation whereby specific chromatin domains are selectively opened along each chromosome. Within each open chromatin domain, a unique battery of gene(s) is availed to tissue-specific and ubiquitous transacting factors that are necessary to initiate transcription. In the absence of an open domain, trans-factor access is denied, and the initiation of transcription cannot proceed. Cell-fate is thus ultimately defined by the unique series of open-potentiated cell-specific chromatin domains. Defining the mechanism that opens chromatin domains is fundamental in understanding how differentiation from stem cells is controlled and whether cell-fate can be modified. A recent examination of the mammalian spermatogenic pathway [Kramer, J.A., McCarrey, J.M, Djakiew, D., Krawetz, S.A., 1998. Differentiation: the selective potentiation of chromatin domains. Development 125, 4749-4755] supports the view that cell fate is mediated by global changes in chromatin conformation. This stride underscores the possibility of moderating differentiation through chromatin conformation. It is likely that gene therapeutics capable of selectively potentiating individual genic domains in populations of differentiating and/or replicating cells that modify cellular phenotype will be developed in the next millennium.

A Matrix Associated Region Localizes the Human SOCS-1 Gene to Chromosome 16p13.13

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3′ end of the human PRM1 → PRM2 → TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3′ of the spermatid-expressed PRM1 → PRM2 → TNP2 domain at position 16p13.13.

Lysyl Oxidase, Cellular Senescence and Tumor Suppression

Replicative senescence may provide a mechanism of tumor suppression and tumor suppressor genes of the extracellular matrix, like lysyl oxidase, may play a role in cellular senescence. To test this hypothesis and determine whether the extracellular matrix may serve as a marker, the steady-state levels of human lysyl oxidase, α-I type III collagen and β-actin transcripts were assessed in various cell lines during in vitro passge. Northern hybridization analysis showed a significant increase in the levels of progeria fibroblast extracellular matrix mRNAs immediately preceding senescence. The levels of these mRNAs were unaffected in age-matched normal fibroblast and fetal fibroblast cell lines.

Temporal expression of the transgenic human protamine gene cluster

OBJECTIVE To ascertain the fidelity of expression of the genes from the transgenic human sperm-specific nuclear packaging protamine-1-->protamine-2-->transition protein-2 (PRM1-->PRM2-->TNP2) locus. DESIGN Controlled human transgene study. SETTING Basic science laboratory. ANIMAL(S) Age-matched transgenic and nontransgenic mice. INTERVENTION(S) Transgenic mice containing the human protamine locus were mated. One testis from each offspring was frozen at -80 degrees C and the other was preserved in formalin. MAIN OUTCOME MEASURE(S) The temporal expression of the human and mouse protamines was evaluated by Northern blot analysis. Orientation of the transgenic locus was determined by Southern blot analysis. Tissue morphology was assessed histologically. RESULT(S) Conservation of transgenic morphology was confirmed. Head-to-tail integration of the PRM1--> PRM2-->TNP2 locus was shown. Temporal expression of the mouse and human protamine genes was maintained in the transgenic state. CONCLUSION(S) These results show that the head-to-tail concatomer of the PRMI-->PRM2-->TNP2 locus contains all the necessary elements for appropriate temporal expression while maintaining testicular structure and function.

Genesis of a novel human sequence from the protamine PRM1 gene

The members of the male haploid expressed protamine 1 (PRM1)-->protamine 2 (PRM2)-->transition protein 2 (TNP2) locus exist as a single, coordinately expressed genic domain. Previous analysis has revealed that the genes within the human PRM1-->PRM2-->TNP2 domain are inter-related, as they share significant sequence similarity at both the nucleotide and amino acid levels. Analysis described here supports the view that a fourth candidate coding region, gene4/Prm3, was derived from PRM1 during the genesis of the PRM1-->PRM2-->TNP2 domain. In some species, gene4 has diverged to a great extent, which can limit its expression.

Matrix-associated regions in haploid expressed domains

1Department of Obstetrics & Gynecology, Wayne State University School of Medicine, 253 CSMC, 275 E. Hancock, Detroit, Michigan 48201, USA 2Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 253 CSMC, 275 E. Hancock, Detroit, Michigan 48201, USA 3C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, 253 CSMC, 275 E. Hancock, Detroit, Michigan 48201, USA