Additional Immune System Functions in the Major Histocompatibility Complex.

Some years ago we conducted an exhaustive search for cDNA fragments from the MHC that might represent products selectively expressed in the immune system. This was based on the hypothesis that the rapid evolution of the MHC would favor the genetic clustering of genes of diverse types whose function would impact on the major role of the MHC in antigen presentation. Among the transcripts detected were 1C7, subsequently shown by others to be a natural killer cell receptor, B144/LST1, and FAT10. B144/LST1 had originally been detected as a murine cDNA fragment by Steinmetz, Olds and their collaborators. We found that human B144/LST1 was expressed in vivo and in vitro in mature dendritic cells. Transfection of B144 into a variety of cell types caused the cells to produce long thin actin based protrusions which we have referred to as cytoneme like filopodia (CLF) (9). Similar effects were seen in stable cell transfectants when B144 expression was induced by removal of tetracycline. Long thin filopodia like structures have been seen by now in a number of cell systems and functions that have been postulated for them include promoting long range cell to cell signaling, and mediating migration of cells towards specific targets We suspect that CLF may be very common cellular features that are readily missed by ordinary microscopy, and that they may lay specific roles in the hematopoietic system. Currently we are evaluating mice in which the B144 gene has been inactivated, and studying proteins that interact with B144.

An additional immune system gene encoded in the MHC , FAT 10, is a homologue of diubiquitin. FAT10 is expressed preferentially in certain cell types such as some lymphoblastoid cell lines, and its expression appears to be tightly regulated at both the transcriptional and post-transcriptional level. Unlike other ubiquitin homologues such as SUMO, FAT10 has been very little studied (17). We are currently investigating mice in which FAT10 has been inactivated, and studying some of the proteins with which FAT10 interacts.

Aging and Related Cellular Phenomena:

Werner syndrome is a progeroid syndrome that causes premature development of a number of manifestations that resemble normal aging. Elucidation of the exact in vivo processes that depend on the WRN protein offers potential insights into the weak points whose malfunction results in some of the common manifestations of aging. Cloning of the WRN gene that is mutated in Werner syndrome patients showed it to be a DNA helicase that is dispensable in mice. Immortalized cell lines lacking WRN have been established. However we find that acute knock-down of WRN in even young primary fibroblasts

leads to rapid accumulation of changes resembling classic cellular senescence as well as intra-nuclear foci resembling those induced by various forms of DNA damage. Currently we are attempting to investigate the relationship of these effects to the cell cycle, as well as the mechanisms mediating these effects and the nature of DNA lesions in WRN knock-down cells.

Our early studies of WRN led to the suggestion that is some relation between telomerase function and WRN. As part of these studies we found that TERT, the catalytic subunit of WRN, my at times be concentrated in the nucleolus, even in the absence of TER, the RNA component of telomerase. TERT contains a complex array of sequences that modulate nucleolar localization, and removal of an N-terminal nucleolar localization signal is associated with markedly diminished activity of the enzyme. In the absence of the nucleolar localization signal TERT protein accumulates but does not associate well with TER and loses activity (3).

Gene Regulation in Hematopoiesis

a. Globin gene regulation.

The human globin gene clusters exhibit remarkably complex regulation, including promoter proximal elements that influence the time of expression of individual genes, and a locus control region (LCR), itself complex, that regulates level of expression of globin genes among other functions. Globin regulation ahs been extensively studied for many years. The accumulated baseline information make the system highly favorable for investigation of novel aspects of gene regulation n cells of higher organisms and a number of fundamental questions remain to be investigated.. K562 cells are a human erythroleukemic cell line that expresses only fetal and small amounts of embryonic globin, and is a frequently used system for studying globin gene regulation. The manner in which the LCR interacts specifically with globin promoters and contributes to chromatin opening and gene expression of these promoters is poorly understood.

We have studied a phylogenetically conserved region in the upstream portion of the promoter for the adult globin gene, and a partially homologous sequence in a portion of the LCR that contributes enhancer activity to the entire LCR. We have identified a protein complex that binds to the upstream promoter region and contains the DNA dependent Swi/SNF like ATPase, HTLF, as a component of this complex. Interestingly cells over-expressing HTLF begin to produce adult globin mRNA (6). We are currently purifying this complex and checking its functional activities.

The partly homologous sequences in the LCR bind other complexes that we have extensively purified. The purified material has very high chromatin remodeling activity and contains an unusual mixture of components found in other chromatin remodeling complexes as well as at least two components not previously associated with chromatin remodeling.

b. Gene expression and regulation during hematopoietic differentiation.

During hematopoiesis there is an ordered differentiation of a stem cell into any one of at least eight major cell types and multiple sub-types of some lineages. Relatively pure populations of normal cells of many types are available because of easy access to blood and the convenience of purification of cells that are or can readily be suspended as single cells. In addition several systems that at least partially reproduce stages of normal development in continuous cell lines are available. We have concentrated on the study of two murine systems, MPPRO promyelocytic cells and EML multipotential cells. MPRO cells differentiate into mature neutrophils on appropriate treatment, and in a coordinate fashion (1,10). EML cells are a multipotential precursor cell line in which there is ongoing commitment of a fraction of the cells to lineages such as myeloid, erythrocytic, and lymphoid cells. As such it offers the potential for in vitro analysis of the process of cell lineage commitment and its modification. In addition we are working with collaborators to study similar processes in normal hematopoietic cells and in mesenchymal stem cells from the bone marrow. Our initial approach has been to use global analyses methods to define patterns of gene expression at the RNA and protein level as a basis for ongoing analyses of the effects of gene knockdown or over-expression on the overall pattern and type of differentiation. We have initiated a substantial effort using RNAi to knock down potential regulatory genes along with parallel studies to introduce normal or mutated genes and to study the effects of these manipulations on differentiation in these several systems.

Methodology of Global Analyses and Functional Genomics:

We have developed a method of highly enriching either perfectly matched DNA duplexes or duplexes containing internal mismatched bases from a pool of double sanded DNA molecules (2). This method has potential for identifying in parallel the polymorphisms in a DNA pool, or the mutations arising by somatic mutation in neoplasias, spontaneously in Mendelian disorders of man, or after ENU mutagenesis in mice. So far, the method has been developed for cDNA but current research is directed towards extending it to total genomic DNA.

In collaboration with the laboratory of Mike Snyder, and as part of the Yale Center of Excellence in Genomics, we are investigating genome wide approaches for analysis of a number of features of human and other mammalian molecular biology. The Genomic tiling arrays consisting of oligonucleotides or DNA fragments spanning all high complexity DNA sequences from large genomic regions are emerging as powerful tools for functional genomics. The Yale Center and Snyder lab have produced tiling arrays of PCR fragments covering selected genomic regions, and we are collaborating in evaluating the use of these for chromatin immunoprecipitation and transcript mapping studies (5,18) In addition we are investigating the feasibility and economics of using non-commercial genomic tiling arrays of oligonucleotides. We hope to use these arrays to map binding sites of transcription factors, three dimensional chromatin architecture, etc. during hematopoietic and mesenchymal stem cell differentiation and development.

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