Missag H. Parseghian

Chromatin Condensing Functions of the Linker Histone C-terminal Domain are mediated by Specific Amino Acid Composition and Intrinsic Protein Disorder †

Linker histones bind to the nucleosomes and linker DNA of chromatin fibers, causing changes in linker DNA structure and stabilization of higher order folded and oligomeric chromatin structures. Linker histones affect chromatin structure acting primarily through their approximately 100-residue C-terminal domain (CTD). We have previously shown that the ability of the linker histone H1 degrees to alter chromatin structure was localized to two discontinuous 24-/25-residue CTD regions (Lu, X., and Hansen, J. C. (2004) J. Biol. Chem. 279, 8701-8707). To determine the biochemical basis for these results, we have characterized chromatin model systems assembled with endogenous mouse somatic H1 isoforms or recombinant H1 degrees CTD mutants in which the primary sequence has been scrambled, the amino acid composition mutated, or the location of various CTD regions swapped. Our results indicate that specific amino acid composition plays a fundamental role in molecular recognition and function by the H1 CTD. Additionally, these experiments support a new molecular model for CTD function and provide a biochemical basis for the redundancy observed in H1 isoform knockout experiments in vivo.

Distribution of somatic H1 subtypes is non‐random on active vs. inactive chromatin II: Distribution in human adult fibroblasts

For nearly twenty years researchers have observed changes in the histone H1 subtype content of tissues as an organism develops into an adult. To better understand the consequences of such changes, immunofractionation of chromatin using previously characterized antibodies specific for human H1 subtypes was employed in the analysis of a fibroblast cell strain derived from a 37‐year‐old individual. DNAs isolated from immunoprecipitates were probed for the existence of a variety of DNA sequences. The results presented lend further support to a previously‐proposed model (Parseghian et al. [ 2000 ] Chromosome Res 8:405‐424) in which transcription of a sequence is accompanied by the selective depletion of subtypes. The data also suggest that there is more total H1 on actively transcribed sequences in these cells as compared to fetal fibroblasts and that there is less difference in the subtype compositions of active genes vs. inactive sequences in this strain. Specifically, the consequences of these changes appear to correlate with the attenuation of the heat shock response in aging fibroblasts. In a broader context, these results could explain why there are reductions in transcription in cells from mature tissue that approach senescence. J. Cell. Biochem. 83: 643–659, 2001.

Fractionation of human H1 subtypes and characterization of a subtype-specific antibody exhibiting non-uniform nuclear staining

Four histone H1 subtypes and H1° were fractionated from human placental nuclei and purified to homogeneity by a combination of Bio-Rex 70 chromatography and reversephase high-performance liquid chromatography (RPHPLC). Polyclonal antibodies were generated in rabbits against one of these subtypes designated H1-3. Antibodies reacted only against this subtype in enzymelinked immunosorbent assays and Western assays; subtype specificity was documented further by Western blotting of cell and nuclear extracts. They crossreacted with monkey H1, but not with H1 from other vertebrates tested. The epitope(s) recognized were mapped by immunoblotting against peptides prepared by cleavage with N-bromosuccinimide (NBS) and α-chymotrypsin; it includes the variant amino-terminal tail of the protein as well as a portion of the globular domain. The antibody stains mitotic chromosomes weakly but uniformly and, unlike antibodies that recognize total H1 which show uniform nuclear staining after indirect immunofluoresence localization, anti-H1-3 exhibits preferential labelling of the nuclear periphery. This non-uniform staining suggests compartmentalization of this subtype which may have functional significance with respect to differential chromatin condensation.

Characterization of a set of antibodies specific for three human histone H1 subtypes

A series of human histone H1 subtype-specific antibodies are described that were generated for localization and functional studies. Since our previous attempts to produce such antibodies against intact subtypes met with limited success, resulting in one antibody against a subtype we have designated H1-3, the approach used in the work presented is based on the production of antibodies against synthetic peptides or peptide fragments encompassing the variant NH2-terminal region of cach protein. Subtype-specific antibodies were obtained against synthetic peptides derived from subtypes designated H1-1 and H1-2 and the NH2-terminal fragment from an N-bromosuccinimide digest of H1–4. Antibody specificities were documented in all cases by enzymelinked immunosorbent and protein immunoblot assays against the purified subtypes as well as immunoblots against whole cell and nuclear extracts. In addition, the in vivo distribution of each antibody was determined by indirect immunofluorescence. H1-1 appears to be distributed in parallel with DNA concentration, similar to the results with an antibody that recognizes all subtypes, However, H1–2 and H1–4 are non-uniformly distributed, exhibiting similar punctate staining patterns. The staining patterns described are different from the pattern desciribed for the distribution of H1–3, suggesting that several subtypes are concentrated in distinct regions of the nucleus and, therefore, may be associated with distinct regions of the genome.

Evicting hitchhiker antigens from purified antibodies

Antibodies that target common cellular structures may have a propensity to bind those very same antigens as they become exposed in dead or dying cells during production in a bioreactor. Those tendencies can be accentuated if the targeted epitope is highly conserved across species. While attention to contaminants such as endotoxin, viral particles, cellular DNA and even prions has grown coincident with the emergence of the monoclonal antibody industry, it is surprising how little attention has been focused on hitchhiker antigens that may co-elute while bound to the supposedly pure antibody. In this case study, we will focus on anti-histone antibodies and the measures we have taken to eliminate stowaways, such as histone-DNA complexes. These simple measures include the addition of a quartenary amine guard column to the protein A, adjusting the ionic strength of the cell culture supernatant to 400 mM sodium chloride, and establishing a mobile phase gradient from 400 mM to 2M during protein A chromatography. Initially adjusting the cell culture to 600 mM can compromise the quartenary amine guard column. Also, we demonstrate the applicability of these techniques in both the R&D lab and the manufacturing plant, particularly in improving the apparent potency of antibodies destined for the clinic. Given the prominence of anti-histone antibodies in chromatin immunoprecipitation (ChIP), the implications of hitchhiker antigens interferring with the results of an experiment are far-reaching, indeed, we detect them in some popularly used antibodies. Moreover, a wide variety of monoclonals that may target antigens expressed by the producer cell line may face similar problems, resulting in a decreased production yield, as well as a diminished apparent binding potency.

In-Line Radiolabeling: A Novel Continuous-Flow System for Commercial-Scale Protein Labeling

A key limitation in developing radiotherapeutic proteins is the expense of manufacturing the drug in small batches using traditional reaction vessels. Removing limitations on the quantity of protein labeled at any one time significantly decreases the cost of production, and nowhere is the need for cost-effective radiotherapeutics more acute than in the treatment of cancer. Methods: We describe a novel method that can rapidly radiolabel, theoretically, unlimited amounts of protein, without causing significant damage to binding potency or structural integrity. Our process controls the reaction rate for the isotope and reactants as they simultaneously flow through a reaction tube. Results: We have demonstrated proof of principle by labeling nearly a gram of antibody with 481 GBq (13 Ci) of 131I during a single 30-min reaction run. Conclusion: Simple to construct, our system is already used to manufacture a radiolabeled antibody, both in the United States and in India, as part of clinical trials to treat glioblastoma multiforme. Modified, this system may be also applicable for nonradioactive labeling.

Clinical update: Treatment of glioblastoma multiforme with radiolabeled antibodies that target tumor necrosis

Glioblastoma multiforme (GBM) is one of the most intractable cancers in humans, and yet, in the past decade, incremental advances in the treatment of brain tumors have begun to suggest that effective therapies may be on the hori- zon. Here we review the latest treatments available to patients and focus on a promising radiotherapeutic strategy that em- ploys the isotope 131 Iodine conjugated to an antibody that binds the necrotic core found in all solid tumors. Historically, GBM patients who relapse have a median survival time of no more than 24 weeks; however, the Tumor Necrosis Therapy discussed here has already provided a good quality of life for several patients years beyond the historical median survival time. The cases of two long-term survivors are reviewed, and data are presented to show that initial post-treatment as- sessments of tumor progression actually turned out to be tumor necrosis and inflammation. Given the current lack of im- aging modalities that can distinguish between tumor progression and pseudoprogression, these cases further highlight the challenges faced by physicians in differentiating disease progression or recurrence from necrosis.