Mohammad Salehi Ph.D Embryologist
Mohammad Salehi Ph.D Embryologist
Chromatin structure in somatic cell • DNA• Proteins
• Histones: rich of lys & arg • H1, H2A, H2B, H3, H4
• Non Histones• Acidic proteins
• Hertons • Enzymes
• Topoisomerase ।,॥• Polymerases• Helicase
• Neutral proteins• Scarcely RNA
DNA structure Nucleotide
Base (A,T,C,G) Pentose sugar (deoxy
ribose) Phosphoric acid
B DNA
Organization of chromatin structure
• First level• Nucleosome: cylindrical shape ( diameter: 10
nm , height: 6nm) • Core particle contains an octamer of 2 each
of the core histones (H2A, H2B, H3 and H4) • 146 bp of DNA wrapped 1.75 turns
• 10 nm fiber: Nucleosome + linker DNA (0-50bp)
Nucleosome
nucleosome- building blocks of chromosomes
H2A: redH2B: yellowH3: purpleH4: green
Histones are small, positively-charged proteins
The nucleosome has an approximate twofold axis of symmetry
Higher-order chromatin structure
H binds to linker DNA at one end ofThe nucleosome and the central DNA helix
The core Histone N-terminal tails are required for the formation of the 30-nm fiber
The tail of H2A, H3 and H4 interact with adjacent nucleosome
Organization of chromatin structure
• Second level• 30 nm fiber
• Solenoid model• Zigzag (crossed linker)
model• Nucleomer (super bead)
model
• Third level• Super coil
The addition of H1 leads to more compact nucleosomal DNA
Without H1
30-nm fiber
Superhelix, 6 nucleosome per turn, supported by EM and X-ray studies
Based on zigzag pattern upon H1 addition, requires linker DNA to pass through central axis,
Higher compaction of DNA involves large loops ofnucleosomal DNA
Nuclear scaffold (Topo II, SMC)
How histones chaperones facilitate the assembly of nucleosomeduring DNA replication
(sliding clamp)
Chromatin organization in sperm
During spermatogenesis spermatogonia undergo modification to produce spermatozoa that include:
• Mitosis (spermatocytogenesis)• Meiosis• Spermiogenesis
• Golgi phase• Cap phase• Acrosomal phase (chromatin condensation)• Maturation phase
Causes of condensation in sperm chromatin
• Reduction of size • Facilitate of sperm penetration into oocyte• Protection of genome from physical,
chemical and biological damages• Reprogram of paternal genome• Coordination of cell cycle between sperm
and oocyte
Protein replacement in spermiogenesis
Transitional Proteins
Somatic H
istones
Testicular Histones
Protamines
Increase basic property
Testicular Histones• Absence of H1• H2A
• H2AZ • H2AX
• H3 and H4 (hyperacetylation)• 15-20% remains in mature sperm
Mechanism of replacement
• Histones acetylation(H4)
• H2AX Phosphorylation
• Ubiquitination
Transitional proteinsType of protein MW (KDa) Amino acid
lengthProperties
HPI1(TP1) 6 54 Rich of arg, lys, hisDNA destabilization and promote of repair
HPI2(TP2) 13 137 Rich of cys, arg, lysBond to CG and cessation transcriptional activity
HPS1 N/A 69 P2 precursor
HPS2 N/A 66 P2 precursor
ProtaminesType of Protamine
MW (Da) Amino acid length
Properties
HP1 6692.2 50 Rich of arg, cys
HP2 7652.4 57 Rich of his
HP3 N/A 54 Rich of arg, cys, his
HP4 N/A 58 Rich of arg, cys, his
Amino acid sequence of P1&P2
Human P2 and P1
Bovine P1Anchoring domains
His, Arg, Arg sequences
P2 family: zinc finger proteins• Contain cys2/his2 motif• Inhibition of
transcription at the end of spermiogenesis
• Stabilization of sperm chromatin by zinc
• P2 family possibly attached to major groove of DNA
Comparison P1 and P2 • P1/P2 ratio= 0.98 ± 0.12• P1 rich of cys• P2 rich of his and few cys compare to P1• P2 is more basic • P2 has more affinity to DNA
Mechanism of condensation• In bull and fish P1 attached to major groove
of DNA• Anchoring domains attached to major groove• After the synthesis of protamine, and before its
deposition on DNA, the serine and threonine residues in protamine are phosphorylated
• After attachment, this residues dephosphorylated
• Formation of intra and inter protamine disulfide bond
Mechanism of condensation• In humans and other mammalian which
have two or more protamine there are contradictory evidence:
• P1 & P2 lie down in major groove• P1 & P2 attached to both minor and major
groove• P1 & P2 attached to external surface of the
DNA chain
Evaluation of nuclear maturity • Chromatin decondensation induced in vitro • DNA staining after chromatin denaturation• Aniline blue staining• Assessment of thiol- disulfide status of nuclei• Ultra structural examination• Biochemical analysis• Comet assay• TUNEL assay• Sperm chromatin dispersion test (SCD test)