METHYLATION OF HISTONE H4 AT

ARGININE 3 FACILITATING
TRANSCRIPTIONAL ACTIVATION BY
NUCLEAR HORMONE RECEPTOR

Danny Monterroso
 Basis of Paper
 Core histone tail modification are important in
chromatin formation/function
 A type of core histone tail modification is
methylation
 Understanding the function of histone
methylation by finding the responsible
enzymes
 Transcriptional Regulation
 Change in gene expression levels by altering
transcription rates
 Identify enzymes
 Nuclear proteins from HeLa cells were
separated into nuclear extract and nuclear
pellet
 Fractionation with DEAE52 and phosphate
cellulose P11 columns.
 Results
 Methyltransferase
activities with
specific targets for
H3 and H4 were
identified.
 Fraction 14 has the
highest concentration
 Silver staining of SDS-
PAGE with column
fractions showed a
polypeptide of 42 kD
eluted with the
enzymatic activity as
well.
 To confirm, gel
filtration superose-200
was used in Figure 1C
 PMRT1  330 kD with
confirmation
 PRMT1 and Lys 20
 Lys-20 had consistently been the one primarily
from H4 to be methylated in vivo.
 Core histone octamers were methylated to
determine if PRMT1 methylated H4 on Lys-20
 Methylated H4 was recovered and cross-
sequenced, and released amino acid derivates
were collected; revealing Arg 3 (not Lys 20)
was the major methylation site.
 Arg 3
 To figure out if Arg 3
methylation occurs in vivo,
antibodies against an Arg 3
methylated H4 amino-
terminal peptide were made.
 Figure 2A shows antibodies
against Arg 3 reacted
strongly with PRMT1 H4,
but not recomb. H4 in E. coli.
 Same antibody recognized
H4 made from HeLa cells in
2B, and shows PRMT1
increases Arg 3 methylation.
 Confirmation of PRMT1-Arg 3 causal
relationship
 To test the actual
relationship of PRMT1 and
Arg 3, core histones from
PRMT1+/+ and PRMT1-/- ES
cells were
purified/compared.
 Past results of Ser 10 made
Arg 3 methylation tests to
see for possible acetylation
interference of H4 tails.
 PRMT1-methylated H4 and p300
 Figure 3B show that
PRMT1-methylated H4 is a
better substrate for p300
when compared with
unmethylated H4.
 K8 and K12 acetylation is
facilitated by Arg3
methylation, but has little
affect on K5/K16.
 Purification of hyper- and hypo-
acetylation core histone HeLa cells
 Histones were
methylated then resolved
through TAU gel,
Coomassie brilliant blue
stain and
autoradiography.
 Determine whether Lys
inhibits H4 methylation
by PRMT1, HeLa cells
were treated with TSA.
 Conclusions
 Arg 3, not Lys 20, is methylated by PRMT1
 Arg 3 facilitates acetylation of H4 tails by p300
 Original methylation site Arg 3 of H4 by PRMT
1
 Arg 3 methylation plays a crucial role in
transcriptional regulation.
 Sources
 Wang Hengbin, et al. Methylation of Histone
H4 at Arginine 3 Facilitating Transcriptional
Activation by Nuclear Hormone Receptor.
Dept of Biochemistry and Biophysics, UNC.
Science 853-855 2001.