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mori PcG proteins regulate BmASNS expression at the appropriate time during the cell cycle

BmC/ebp mediated activation since BmPHO and BmSCM RNAi in the presence of

BmC/ebp cause similar up-regulation of the promoter activity (Fig. 38 and Fig. 41).

To further estimate a relative power balance between the two transcriptional

regulators, I performed PcG genes RNAi under the enforced expression of BmC/ebp.

Overexpression of BmC/ebp up-regulated the BmASNS promoter activity on the all

deletion constructs (comparing Fig. 49A with Fig. 48). Interestingly, the knockdown of

BmPHO or BmSCM still significantly increased transcription on the p4974 and p3614

under the BmC/ebp overexpression, slightly increased on the p2285, but not on the

p1056 construct. These results showed that the BmC/ebp transactivator can basically

compete with PcG proteins in the presence of the CpG island region. This critical role

of the CpG island was also supported by the similar experiment on the other two

deletion constructs, pASNS∆YY1 and pASNS∆YY1/CpG (Fig. 49B).

B. mori PcG proteins regulate BmASNS expression at the appropriate time during

Although the results described above demonstrated a fact that PcG system is

exactly involved in the repression of BmASNS expression, it was still puzzled that, why

PcG complexes are able to regulate a “housekeeping” gene that differs from other

well-documented PcG targets such as Hox genes in D. melanogaster as well as in

mammals, and it might be not the canonical fashion regulated by PcG system on the

BmASNS gene.

Following the clue that ASNS gene can be induced to express at G1 phase or

before DNA replication in human cells (Greco et al., 1989), I examined the expression

profile of BmASNS gene during the cell cycle of silkworm cells. Because there was no

effective method to synchronize cell progression in silkworm cells, I developed a novel

strategy that took advantage of cell cycle-regulated factors with the ability of inducing

cells arrest at different phases upon RNAi treatment. Here, the knockdown efficiency of

each gene by the specific dsRNA in the BmN4-SID1 cells was evaluated by RT-PCR

(Fig. 50A). I further confirmed, using flow cytometry analysis, that cells lacking

BmMYC gene primarily accumulated at the G1 phase of the cell cycle, and BmCDT1

affect entry of cells into the G2 phase. We also observed that the loss of BmCDK1

would arrest cells at the G2/M phase and of BmCDC27 led to an effective arrest at the

metaphase (Fig. 50B). Based on these arrested cells, semi-quantitative RT-PCR was

used to detect the expression pattern of BmASNS gene during the cell cycle progression.

As shown in Fig. 51, the expression levels of BmASNS were very low at the M phase

(BmCDC27 knockdown), and after cell division, the expression was up-regulated,

particularly at the late-G1 phase (BmCDT1 knockdown), consistent with the pattern in

human cells (Greco et al., 1989). These observations confirmed that the expression

profile of BmASNS is distinct among different phases of the cell cycle, and indicated

that the PcG system allows timely control of BmASNS expression to accommodate the

needs of cells.

To test this possibility, I further performed separately the knockdown of

BmPHO, BmSCM, or BmC/EBP in the knockdown cells from the foregoing experiment,

respectively. As shown in Fig. 52, when cells were arrested at the G1 or S phase

(BmMYC, BmCDT1, or BmRNRS dsRNAs-treated cells), the concomitant

down-regulation of BmPHO or BmSCM did not greatly change the expression of

BmASNS gene, whereas significant increases of BmASNS levels in BmPHO or BmSCM

-depleted cells were observed in the cells arrested at the G2/M phase (BmCDK1 or

BmCDC27 dsRNAs-treated cells). On the other hand, the further loss of BmC/EBP

caused the significant decreases of BmASNS transcription by the BmMYC, BmCDT1, or

BmRNRS RNAi treatment. All together, these revealed that PcG complexes cooperate

with BmC/ebp to regulate the BmASNS promoter activity in a cell cycle dependent

manner.

DISCUSSION

In this study, I reported that PcG system directs gene regulation in a given

locus in B. mori and elucidated a new mechanism under the regulation of PcG

complexes on its target gene BmASNS, namely PcG complexes can counteract with

C/ebp transactivator in the specific cell phase of the cell cycle through the cis-regulatory

elements of YY1 binding motifs and CpG island present on the BmASNS promoter.

D. melanogaster PREs containing YY1 motifs for Pho binding serve as a

platform for the binding of Pho protein and other PcG proteins that participate in the

modification of histone and compaction of chromatin in the local region (Muller and

Kassis, 2006). Actually, the YY1 binding sites in the silkworm BmASNS promoter are

also found to be involved in recruiting of the PcG complexes. However, what is

different from the model in D. melanogaster is that the CpG island in BmASNS

promoter could facilitate this repressive effect from PcG proteins through a potential

mechanism of increasing the recruitment amount of PcG proteins or elevating the

well-known observation about the involvement of CpG islands in the regulation of PcG

complexes in mammals (Deaton and Bird, 2011). It remains to be determined whether

such CpG island-mediated regulations are common in insects.

In D. melanogaster and mammals, the PcG system generally controls the

expression of target genes by influencing the status of H3K27 methylation-deposited by

PRC2 complex in their promoter region. ChIP assays in this work revealed a high level

of H3K27me3 in the BmASNS promoter, especially near the CpG island. Moreover, the

H3K27me3 was maximally reduced after down-regulating the expression of the PRC2

components, indicating that the PRC2 complex rather than other complexes contribute

to the tri-methylation of H3K27. Thus, it is reasonable that the formation of condensed

chromatin structure mediated by H3K27me3 will result in a high repressive effect on

the prompter activity, the same as the observation in the depletion of BmESC in Chapter

II. In addition, it is noted that the H3K27me3 can be inherited as epigenetic memory at

the chromosomal locus (Ringrose and Paro, 2004) and may provide a repressive signal

for the subsequent restructuring of PcG system mediated by the PRC1 complex (Cao et

al., 2002). But, because there is no epigenetic memory in the episomal construct newly

introduced into the cells, the present results thus showed that BmPHO or BmSCM

should play more critical role in a creation of new epigenetic memory at the episomal

construct, compared to the chromosomal locus. This hypothesis, however, is not

sufficient to explain why the knockdowns of all PcG genes induce similar effects on the

chromosomal locus. In other words, this observation raises one issue that the repression

of BmASNS may be ascribed to the binding of PcG proteins per se or the established

H3K27me3. The previous research in Chapter II suggested the presence of at least two

types of regulatory complexes, namely the whole PcG system and Pho/Scm complex.

Taken together with the current results, it is apparent that the B. mori PcG system is not

enough to suppress the BmASNS promoter activity without the continuous expression of

BmPho or BmScm protein, suggesting that the complex of BmPho/BmScm will be

required to facilitate this repression from the complete PcG complexes. In particular, it

was also observed that the repressive effects on BmASNS promoter of all PcG proteins

overexpression were similar as that at the episomal constructs. Therefore, all these

findings exposed that the removal of the PcG proteins from this region is seemed to be

gene.

Genome-wide expression analysis in mouse hematopoietic stem cells has

shown that PcG proteins and C/ebp can regulate a large set of genes in a positive or

negative manner (Majewski et al., 2010). In this study, I identified that B. mori PcG

proteins negatively regulate the BmASNS expression by counteracting the transactivator

BmC/ebp. This case of BmASNS gene in B. mori is in agreement with the negative

correlation of PcG proteins and C/ebp on target gene expression in mouse (Majewski et

al., 2010), therefore suggesting a common model that most target genes orchestrated by

PcG complexes may tend to be cooperatively regulated by C/ebp protein. It is worthy of

further investigation to determine the target genes of BmC/ebp in a genome-wide scale

and to analyze their correlations integrating with the microarray data from the

knockdowns of PcG genes.

Importantly, I demonstrated that PcG proteins play roles in the regulation of

BmASNS promoter activity at the specific phase of the cell cycle and confer a model for the dynamic regulation of BmASNS expression-involved by PcG repressors and

BmC/ebp activator, as shown in Fig. 53. According to this model, before cells enter into

the S phase, uptake of various nutrient elements including amino acids is required. To

increase the BmASNS transcription at the late G1 phase, the cells have to remodel the

chromatins around the BmASNS promoter followed the release of PcG complexes and

the increase of the accessibility of promoter to transcriptional activators including

BmC/ebp. I speculated that a small amount of H3K27me3 marks will be maintained in

this locus if not all, since the previous studies have revealed that this modification can

be maintained at sites of DNA replication and even during the cell division (Hansen et

al., 2008), and the remaining H3K27me3 can help PRC1 complex recognize the locus

on BmASNS promoter in the next cell phase. After the completion of S phase, the

expression of BmASNS gene will return back to a low level mediated by the rebinding of

PcG system and/or Pho/Scm complex at the G2 and M phases. The presence of PcG

complexes may in turn increase the levels of H3K27me3 and promote the compaction

of local chromatins, whereas Pho/Scm complex assists the repression by the whole PcG

complexes. The comprehensive impact of this procedure will lead, at a certain extent, to

loss of BmC/ebp binding and then attenuate the promoter activity. Notably, it will

require a balance between the repressor and activator to maintain this expression state.

SUMMARY

In summary, the present study has elucidated a novel epigenetic regulation in

which PcG complexes regulate BmASNS expression involving repressive mark

H3K27me3. The data demonstrated that PcG proteins repress the transcription of

BmASNS gene through recruiting to the putative YY1 binding motifs and CpG island

within the BmASNS promoter. It is therefore tempting to speculate that the YY1 binding

element, as well as the CpG island is sufficient to recruit PcG complexes and

subsequently deposited H3K27me3 to repress target gene expression in B. mori.

Particularly, this study provides important new insights into the mechanism underlying

the dynamic regulation of its target gene by PcG system during the cell cycle.

GENERAL DISCUSSION

Polycomb group (PcG) proteins are involved in chromatin modifications for

maintaining gene repression that play important roles in the regulation of cell cycle

progression, tumorgenesis, chromosome X-inactivation, and genomic imprinting in D.

melanogaster, mammals, and even plants. Although the regulatory mechanisms of PcG

complexes have been well understood right now, the emerging evidence have shown

much more complicated and varied ways in different species. Therefore, the aim of this

work is studied to understand the action mechanism of PcG system in the model insect

B. mori.

I first identified the orthologs of PcG genes in B. mori, and obtained 13

candidates by using the D. melanogaster PcG genes as queries. These proteins possess

various and important domains that provide the possibility of executing multiple

functions for PcG proteins during development. The further comparative analysis of the

insect PcG proteins has revealed the conserved evolution of the PcG proteins in insects.

Similar to the expression pattern of D. melanogaster PcG genes during embryogenesis,

high-level expressions of all the B. mori PcG genes were also observed and this profile

was maintained through day 2 to day 7 of the B. mori embryogenesis, indicating the

crucial roles of PcG genes in this developmental stage.

The cloned 6 PcG genes in this study have shown the conserved cell nuclei

localization and transcriptional repression activity, and also knockdown of each PRC2

component considerably decreased the global levels of H3K27me3 but not of

H3K27me2. All these results confirmed the present identification of PcG proteins in B.

mori.

And then the microarray expression screening was used to identify PcG target

genes in a genome-wide scale based on the knockdown of BmSCE, BmESC, BmPHO, or

BmSCM gene, which represent the distinct PcG complexes. As a result, the expressions

of 29 genes were up-regulated after knocking down these four PcG genes. Particularly,

there is a significant overlap between targets of BmPho (331 out of 524) and BmScm

(331 out of 532), and among these, 190 genes function as regulator factors playing

important roles in development. The further data found that BmPho, as well as BmScm,

revealed that the C-terminus of BmPho containing zinc finger domain is involved in the

interaction between BmPho and BmScm. Moreover, the zinc finger domain in BmPho

contributes to its inhibitory function and ectopic overexpression of BmScm is able to

promote transcriptional repression by Gal4-Pho fusions including BmScm-interacting

domain. Loss of BmPho expression causes relocalization of BmScm into the cytoplasm.

Collectively, I provide evidence of a functional link between BmPho and BmScm, and

propose two PcG-related repression mechanisms requiring only BmPho associated with

BmScm or a whole set of PcG complexes. It would be interesting to examine whether

this case is also present in D. melanogaster or other species, which could enrich our

understanding of the regulatory mechanism of PcG proteins.

Finally I elucidated a specific action mode underlying the PcG regulation on a

discrete gene locus. I demonstrated that PcG proteins and transcription factor C/ebp are

involved in the transcriptional regulation of the BmASNS gene. The cis-regulatory

elements YY1 binding motifs and CpG island located in the BmASNS promoter are

required for the recruitment of PcG complexes and the subsequent deposition of

H3K27me3. RNAi-mediated knockdown of PcG genes led to a significant increase in

BmASNS expression by recruiting activators including BmC/ebp to the promoter.

Interestingly, I found that PcG proteins and BmC/ebp regulate the BmASNS expression

in a cell cycle-dependent manner. It will be necessary to repress the BmASNS

expression at the G2/M phase in the presence of BmC/ebp activator. The data will shed

fresh light on the regulation of ASNS gene in human, which may explain a potential

mechanism for the tumorgenesis mediated by PcG system via the Asns functions.

In conclusion, the present study on the identification and functional

characterization of B. mori PcG complexes has revealed some novel insights into the

regulation mechanisms. These findings will also provide fundamental knowledge useful

for further investigations of PcG functions in B. mori.

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