Year of Publication
Doctor of Philosophy (PhD)
Arts and Sciences
Dr. Jason DeRouchey
During spermiogenesis, somatic chromatin is remodeled and a vast majority (> 90%) of DNA histones are replaced by short arginine-rich peptides called protamines. This compaction is immense, with protamine-DNA self-assembly in sperm chromatin resulting in a final volume roughly 1/6th of a somatic nucleus. This near crystalline organization of the DNA in sperm is thought crucial both for the transport of the paternal genes as well as for the protection of genetic information as sperm chromatin is transcriptionally inactive and all DNA repair mechanisms are shut down.
Chapter 1 will include an overview of the topics discussed in this document, including: sperm chromatin, Sperm chromatin remodeling, DNA damage, and the effect of DNA damage to sperm DNA.
Chapter 2 will contain a brief overview of the techniques used within this study. This includes: Small-angle X-ray Scattering, gel electrophoresis, DNA precipitation assays, and ethidium bromide dissociation assays.
In chapter 3, we will discuss the effect of DNA packaging on the accessibility of free radicals to damage condensed DNA. A variety of polycations were used to condense plasmid DNA in reconstituted samples. After condensation, the DNA-polycation condensates were exposed to 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) for 1 hour, decondensed, and the plasmid DNA examined by gel electrophoresis. By comparing the intensities of the supercoiled, open coiled and linear bands, we were able to identify the presence of single-strand nicks and double-strand breaks in DNA. DNA packaging densities for all polycation-DNA systems were determined by small-angle X-Ray scattering (SAXS). Our results show that for similar length polycations, the amount of oxidative damage scales directly with the DNA packaging with more tightly condensed DNA being damaged less. However, our results also show that DNA damage is also dependent on polycation length, with DNA condensed by shorter polycations being damaged more than DNA condensed with longer polycations even at similar packaging densities.
Protamine has long been thought to play a role in protecting spermatic DNA from damaging agents in vivo. However, the relationship between the hypercondensation of sperm chromatin, the DNA integrity, and the transfer of epigenetic information from sperm to oocyte and potential to alter gene expression in the early embryo are poorly understood. In Chapter 4, we examine how underprotamination affects free radical accessibility and DNA stability in reconstituted sperm chromatin. Specifically, reconstituted salmon protamine- plasmid DNA condensates (polyplexes) were formed at precise protamine/DNA ratios and subsequently subjected to exposure to AAPH free radicals. Agarose gel electrophoresis was then used to assess DNA damage by observing topology alternations in the decondensed polyplexes. FPG-DNA glycosylase has also been used to more accurately determine oxidative damage beyond just nicks and double-strand breaks in the various condensed states. We show that higher levels of protamination correlate to greater levels of protection to the DNA from oxidative damage up until full charge compensation. Furthermore, we also demonstrate that poorly compacted chromatin could be recovered by the introduction of small cationic peptides in underprotaminated condensates as well as actual sperm nuclei. SAXS studies were performed to show that the introduction of cationic peptides resulted in tighter DNA packaging densities in the underprotaminated sperm chromatin.
In Chapter 5, we examine the role of disulfide bonds on DNA packaging in mammalian sperm chromatin. Mammalian protamine, unlike fish, are known to have cysteine residues capable of forming inter- and intra-protamine disulfide bonds. In bull, prior work had shown evidence for the formation of a unique hairpin secondary structure due to the folding of the ends of the protamine molecule by intramolecular disulfide linkages. Between folds is an arginine-rich region known as the DNA binding region. The DNA binding region has a local arginine fraction (~60-75%) that is much higher than the arginine fraction within the full bull protamine sequence (~50%). Previous work by the DeRouchey lab has shown that the percent arginine was crucial for DNA condensation in small arginine-rich peptides. We hypothesize that the fraction of arginine is also critical to DNA remodeling in sperm chromatin. SAXS studies showed that disulfide bond reduction resulted in complete decondensation of bull sperm nuclei. Here, we have used cysteine alkylation chemistry to add neutral or charged functional groups to the protamine cysteine, thereby inhibiting the formation of these disulfide bonds. This chemistry both prevents the formation of the hairpin as well as modifies the overall charge of the protamine. Through ethidium bromide exclusion assays, we measured binding of these altered protamines to calf thymus DNA and determined that a percent cationic charge of above 50% is necessary for the protamine to effectively condense DNA. In addition, we show that DNA condensation of bull protamine with the hairpin is nearly identical to piscine protamines which have no disulfide linkages but a net arginine fraction of 60-75%. Upon disruption of the hairpin, however, complete condensation does not occur despite a net charge on the protamine of +26.
Digital Object Identifier (DOI)
This work was supported by NSF MCB-1453168.
Kirchhoff, Daniel, "ILLUMINATING DNA PACKAGING IN SPERM CHROMATIN: HOW POLYCATION LENGTHS, UNDERPROTAMINATION AND DISULFIDE LINKAGES ALTERS DNA CONDENSATION AND STABILITY" (2019). Theses and Dissertations--Chemistry. 112.