Histone modifying enzyme, HAT and HDAC have been supposed to play an important role

1
(No Transcript)
2
Introduction ? Histone modifying enzyme, HAT and
HDAC have been supposed to play an important role
in transcriptional regulation by altering
chromatin structure.
3
? Hyperacetylation of chromatin is generally
associated with transcription activation, whereas
hypoacetylation of chromatin is associated with
transcriptional repression
4
? TGF-ß has been implicated in a wide variety of
cellular processes regulation cell cycle,
cell differentiation, and extracellular Matrix
synthesis ? TGF-ß primarily exert its
biological effect through interactions with TGF-ß
type II receptor(TßRII)
5
? Inactivation of TßRII contiributes to malignant
transformation at an early step of tumorgenesis.
many human cancer cell lines express normal
TßRII and downstream signaling intermediates, but
express significantly low or undetectable
levels of TßRII mRNA ? Transcriptional repression
of TßRII gene might be more common mechnism
leading to TGF-ß resistance
6
? MS-275 Synthetic inhibitor of HDAC
Induction the accumulation of acetylated
histones(H3, H4) in the chromatin of the TßRII
gene (but not TßRI) Induction TßRII mRNA but
TßRI mRNA in human breast cancer cell line ? PCAF
a protein with an intrinsic HAT
activity.(transcription coactivator)
7
Fig. 1. Activation of TßRII promoter by MS-275,
TSA and NaBu in MCF-7 cell. ? MS-275 cell
human breast cancer cell
line ? Transient transfection
TßRII/luciferase reporter cinstruct
pRIIP-219/36-luc Control vector
pGL3-basic ? MS-275, HDAC inhibitor, induces
TßRII gene expression at a transcription level
0.5µM 0.3µM 2mM 24hr
8
Fig. 2. Activation of stably integrated TßRII
promoter by MS-275 ? MS-275 treatment 0.5 µM,
25 hr
? MS-275 has the same effect in stable cell line
expressing the TßRII promoter/luciferase gene as
in cells expressing the transiently transfected
TßRII promoter
9
Fig. 3. Activation of TßRII promoter deletion
constructs by MS-275 in MCF-7 and ZR-75 cells. ?
Presence of an element responsible for the
induction within the region from 219 to 172. ?
From 100 to 47 contains a major element
required for the induction by MS-275
10
Fig. 4. Identification of the CCAAT-box element
required for MS-275 induction of TßRII
promoter. ? M3 and M4 contain mutation of
inverted CCAAT box(-82 to 78) ? CCAAT box
v-src-mediated induction of the TßRII promoter ?
CCAAT box plays a critical role in the induction
of the TßRII promoter by the HDAC inhibitor,
MS-275.
11
Fig. 5. Identification of the nuclear proteins
which binds to the MS- 275-responsive element of
the human TßRII promoter
? Complex a, b, and c is specific binding of
protein. ? M3 mutation inverted CCAAT
consensus seuquence
12
Fig. 6. Binding of NF-Y to the CCAAT box
mediates MS-275 induction. ? ESE an ERT
promoter specific element ? NF-Y
Binding to the CCAAT box (prvious report) A
complex composed of the three subunit
NF-YA(CBP-B), NF-YB(CBP-A) and BF-YC(CBP-C)
? Complex A represents NF-Y protein bound to the
CCAAT boxes of the TßRII promoter in human
breast cell line and binding activity of NF-Y
protein is not affected by MS-275
13
Fig. 7. PCAF is recruited to the CCAAT box
by treatment with either MS-275 or TSA. ? NF-Y
protein is connected with histone
acetyltransferase activity. physical
associaction with the related histone
acetyltransferase (hGCN5 and PCAF) in vivo ?
TSA increased the activity of NF-Y- Dependent
promoter. ? DNA affinity-pull down assay DNA
conjugated magnetic bead Nuclear extract
? Precipitation by
magnetic plate
? Resuspension and reprecipitation by centri.
? Elution and
western blotting
14
? Activation of TßRII promoter by an inhibitor of
HDAC is due to the increase of HAT activity by
recruiting PCAF to NF-Y tethered to the TßRII
promoter in human breast cancer cell
line. Discussion ? The induction of TßRII gene
by MS-275 require an intact CCAAT box and its
cognate binding factor, the NF-Y complex. ?
When cell are treated with an HDAC inhibitor,
PCAF with histone acetyltransferase activity, is
recruited into the NF-Y complex, increasing the
activity of the TßRII promoter. ? A novel
mechanism for the activation of the TßRII
promoter by HDAC inhibitor 1st TßRII promoter
is repressed by compact chromatin structure,
which is maintained by increased HDAC
activity. 2rd NF-YB/C contain a histone-like
motif and interact in a nucleosome-like structre.