Zelda's Effect on Gene Expression in the Early Development of

Drosophila Embryo

Jing Wang

School of Arts, Fordham University, New York, NY, 10023, U.S.A.

Keywords: Zelda, Drosophila Embryos, Transcription Factors, Pair-Rule Genes, Gap Genes.

Abstract: Zinc-finger protein Zelda (Zld) is thought to play an essential role in the development of early-stage

Drosophila embryos, and this paper takes a step further by confirming the Zld’s effect on Class I & II gene

expression, and exploring the synergistic effects of Zld and other transcription factors in gene expression. The

primary methods used in this paper are Zelda-binding pattern and JASPAR analysis results. It was found that

Zld played a direct and indirect, decisive and non-decisive role in Class I and II gene, respectively. Besides

Zld, this paper spotted that the gap proteins derived from gap genes were an key transcription factor in the

expression of the pair-rule genes, a subdivision of the Class II genes. There is also evidence shows that sloppy

paired 1 might be the enhancer of sloppy paired 2. On the basis of previous studies, this work studied the

effects of Zld and other transcription factors on the expression of different types of genes in more detail.

Directions for future research were discussed.

1 INTRODUCTION

In recent years, several studies have explored the

critical role of the zinc-finger protein Zelda (Zld) in

early embryonic drosophila. (Liang, Nien, Liu,

Metzstein, Kirov, Rushlow 2008) confirmed Zld as a

key activator in the early zygotic genome; (Nien,

Liang, Butcher, Sun, Fu, Gocha, Kirov, Manak,

Rushlow 2011) investigated how Zld affects the

timing mechanism of the development of early genes

(Fu, Nien, Liang, Rushlow 2014); demonstrated that

Zld is a predictor of enhancer activity and the co-

coordinate to regulate gene expression. However, the

synergistic effects of Zld and other transcription

factors in the regulation of gene expression in the

preliminary stage still need to be further explored.

For narrowing such a gap, this paper will use

Zelda-binding patterns and JASPAR analysis results

to summarize the effects of Zld on Class I & II genes

and explore whether Zld coordinates with other

transcription factors to influence the gene expression.

In this paper, we defined the Class I gene as the

ubiquitous gene expressed throughout the embryo at

an earlier stage of development. Class II gene, also

known as patterning gene, is expressed in some

regions of embryos in the later stage of development

according to a specific pattern.

We utilized Kuk (CG575) and pairing-rule genes

Slp 1&2 (CG6738 & CG2939) as Class I and II gene

representatives, respectively. Our research showed

that in Class I genes, Zld directly binds to the

promoter to activate its expression, while in Class II

gene, Zld binds to the enhancer to affect certain

expression levels. We also spotted other transcription

factors, gap proteins, that control Class II genes'

expression patterns.

2 METHODS

2.1 Integrated Genome Browser (IGB)

We used Integrated Genome Browser (IGB) to put

several experimental data in order based on

Drosophila melanogaster genes. Wildtype RNA

polymerase expression in Cycle 12 was used as the

baseline reference; wildtype RNA polymerase

expression in Cycle 13 and RNA polymerase

expression in Cycle 13 when Zld knocked out were

used to compare the effect of Zld on gene expression;

wildtype ChIP-seq of Zld and TAGteam

(CAGGTAG) site were used to confirm the Zld

binding pattern. This step enables us to obtain a

620

Wang, J.

Zelda’s Effect on Gene Expression in the Early Development of Drosophila Embryo.

DOI: 10.5220/0011249800003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 620-625

ISBN: 978-989-758-595-1

preliminary inference about Zld binding patterns

toward the selected genes for conducting further steps.

2.2 JASPAR

JASPAR was used to further affirm Zld’s effect on

gene expression and predict whether other

transcription factors were involved in gene

expression (JASPAR 2020). We first got the Zld

binding peak coordinate from the IGB and used

intercept ± 200bp of it as the region to obtain the

FASTA-formatted targeted gene sequence in

Biotechnology Information (NCBI) (National Center

for Biotechnology Information) database. Based on

the criteria with a relative profile score threshold of

80%, JASPAR scanned all transcription factors that

have the possibility to bind to the given gene

sequence and arranges them by correlation.

2.3 FlyBase & Berkeley Drosophila

Genome Project (BDGP)

FlyBase and Berkeley Drosophila Genome Project

(BDGP) (BDGP 2021) are both information libraries

that can provide data to consolidate our hypothesis.

FlyBase database contains information about genes

and transcription factors, while BDGP contains gene

expression images at different stages.

3 RESULTS & DISCUSSION

3.1 Class I Genes (Ubiquitous Genes)

Kugelkern (kuk, CG5175). “[kuk] encodes a nuclear

envelope protein required for nuclear elongation

during cellularization.” (FlyBase Homepage) And it

can be drawn from Figure 1 that kuk is a typical class

I gene as it is expressed throughout the whole

embryo.

Figure 1: Expression Pattern Image for kuk from BDGP.

As shown in Figure 2, the highest Zld binding

peak in the 4th track corresponds to the TAGteam site

(CAGGTAG) in the 5th track. We further used

JASPAR to scan ±200bp near this site, as shown in

table 1, confirming that there was indeed a strong Zld

binding site with a high score of 13.82. Then, by

comparing the kuk expression with or without Zld,

we see that a considerable amount of RNA expression

is demonstrated in wildtype case (the second track),

while the expression level witnesses a huge decrease

when Zld was knocked out (the third track). Such a

phenomenon suggests that Zelda is a key

determinator for the expression of kuk.

Note. RNA polymerase expression in Cycle 12 (the first green track); wildtype RNA polymerase expression in Cycle 13 (the

second pink track); RNA polymerase expression in Cycle 13 when Zld knocked out (the third blue track); wildtype ChIP-seq

of Zld (4th red track) and TAGteam site (5th brown track). The previous four tracks are on the same scale from 0 to 300.

Figure 2: Snapshot of Gene Kuk From IGB.

Zelda’s Effect on Gene Expression in the Early Development of Drosophila Embryo

621

Table 1 JASPAR –kuk–vfl–Analysis Result

Matrix ID Name Score Relative score Sequence ID Start

Stran

Predicted

sequence

MA1462.1 vfl

13.8206

0.993095999606

NT_033777.3:17082105-

17082505

197 208 +

CGGCAGGTAG

MA1462.1 vfl

12.0672

0.958080363668

NT_033777.3:17082105-

17082505

221 232 -

TTGCAGGTAC

Note. Vfl, as known as Zld; The higher the score, the higher the affinity that the transcription factors bind to the gene sequence.

3.2 Class 2 Genes (Patterning Genes)

This research selected sloppy paired 1 (slp1) &

sloppy paired 2 (slp2) / CG16738 & CG2939 as the

Class 2 gene for analysis because they accord with

the Class 2 gene’s criterion of being expressed in a

certain area of the embryo at a later stage of

development (demonstrated in Figure 3). Figure 3

also reveals the similar expression pattern that slp1

and slp2 share, that is, start from the head and

gradually extend to the whole embryo in strips with

intervals. It shows the slp1 and slp2’s role in the

process of establishing body segments as pair-rule

genes.

By comparing the second and third tracks of

Figure 4, we found that after Zld was knocked out,

the gene expression of slp1 experienced a moderate

decline, while the gene expression of slp2 did not

change significantly. In the fourth column, several

Zld binding peaks in this gene segment were

displayed, and JASPAR analysis confirmed that Zld

indeed has high scores in these peaks both in slp1 &

slp2 (Table 2). Therefore, we speculated that Zld

could play an influential but not decisive role in the

expression of Class 2 genes, and other transcription

factors are of more significant impact on their

expression.

Note. Drosophila embryos are in the developmental stage

4-6.

Figure 3: Expression Pattern Image for slp1 & slp2 from

BDGP.

Note. Track contents are the same as Figure 2.

Figure 4: Snapshot of gene slp1 & slp 2 from IGB.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

622

Table 2: JASPAR -slp1 & slp 2 -vfl- Analysis Results Note. The number in the Peak column stands for Zld binding peak in

the 4

track of Figure 3.

Gene Matrix ID

Score Relative score Sequence ID Start End Strand Predicted sequence Peak

Slp2 MA1462.1 vfl 13.2734

0.982167314

844

NT_033779.5:3833900-

3834100

176 187 + CGGCAGGTAGCG 1

Slp1 MA1462.1 vfl 12.2371

0.961472452

634

NT_033779.5:3825455-

3825655

79 90 - CATCAGGTAGTT 3

Slp1 MA1462.1 vfl 12.0538

0.957812957

086

NT_033779.5:3822050-

3822450

175 186 - CTTCAGGTAGTG 1

Slp1 MA1462.1 vfl 11.035

0.937467450

787

NT_033779.5:3822050-

3822450

144 155 - ATCCAGGTAAGA 1

Slp2 MA1462.1 vfl 10.8576

0.933924242

155

NT_033779.5:3835455-

3835655

83 94 + GCTCAGGTAAAA 2

Slp1 MA1462.1 vfl 10.4841

0.926465843

882

NT_033779.5:3824600-

3825000

185 196 - ACTCAGGTAATC 2

Slp2 MA1462.1 vfl 10.0659

0.918115258

717

NT_033779.5:3833900-

3834100

40 51 + GCGTAGGTAGGA 1

Table 3: JASPAR -slp1 & slp 2 -gap genes- Analysis Results.

Gene Matrix ID

Score Relative score Sequence ID Start End Strand Predicted sequence Peak

Slp2

MA0452.

Kr 14.616 0.937574067021

NT_033779.5:3833900-

3834100

149 162 + CTTAACCCCTTCAG 1

Slp1

MA0452.

Kr 14.5752 0.93695773075

NT_033779.5:3824600-

3825000

172 185 + TTTAACCCCTTCGG 2

Slp1

MA0452.

Kr 13.7451 0.966483513258

NT_033779.5:3824600-

3825000

174 184 - CGAAGGGGTTA 2

Slp2

MA0452.

Kr 13.5852 0.922015290106

NT_033779.5:3833900-

3834100

1 14 + CTTAACTCTTTCGA 1

Slp1

MA0049.

hb 12.8235 1.0000000052

NT_033779.5:3822050-

3822450

388 397 + GCATAAAAAA 1

Slp2

MA0452.

Kr 12.4301 0.933338080611

NT_033779.5:3833900-

3834100

151 161 - TGAAGGGGTTA 1

Slp2

MA0452.

Kr 11.79 0.917205123065

NT_033779.5:3833900-

3834100

3 13 - CGAAAGAGTTA 1

Slp1

MA0459.

tll 11.6933 0.887199901339

NT_033779.5:3822050-

3822450

205 214 + AAAAGTGAAA 1

Slp2

MA0049.

hb 11.2386 0.951168571865

NT_033779.5:3835455-

3835655

68 77 - TCATAAAAAA 2

Slp1

MA0452.

Kr 10.833 0.880472403084

NT_033779.5:3824600-

3825000

71 84 - GGCAATCCTTTTGG 2

In addition, it was also found from JASPAR

analysis (Table 3) that both slp1 and slp2’s peaks

have high scores of gap protein, e.g., Kruppel (Kr),

Hunchback (hb), and Tailless (tll). According to

Griffiths et al., Kr and hb both are regulators, but

repressor and activator, respectively, jointly control

the expression of the pair-rule gene. Their differences

in concentration at the embryo’s position control each

pair-rule stripe formation (Griffiths, Doebley,

Peichel, Wassarman 2020). Our data reaffirm the

above findings and identify one more gap protein, tll,

as the regulator for forming embryonic stripe

formation.

Other than gap genes, another high score gene

repeated shows up in the peak of slp1 from JASPAR

analysis results (Table 4), namely defective

proventriculus (dve), which is considered as a

transcriptional repressor that involves in

Zelda’s Effect on Gene Expression in the Early Development of Drosophila Embryo

623

developmental patterning (FlyBase Homepage). We

speculate that dve has the same function as the Kr to

control the slp1 gene expression. The data presented

in this paper support such a view, but specifically,

how dve influences the embryonic stripe formation

remains to be determined by further studies.

We also deduced the relationship between slp1

and slp2 through IGB graphic and JASPAR analysis.

Firstly, from the zoom-out snapshot of the two genes

(Figure 4), we found that slp1 and slp2 appeared in

pairs, and slp1 appeared earlier than slp2. Second,

slp1 protein shows high scores in both peaks of slp2

(Table 5). Both phenomena are suggesting that slp1

is an enhancer of slp2. However, this is only

speculation based on the data. A control experiment

should be carried out to compare the expression of

slp2 with or knocking out slp1 to determine the role

of slp1 in slp2’s expression.

Table 4 JASPAR - slp 1 - dve- Analysis Results

Matrix ID

Score Relative score Sequence ID Start End

Strand

Predicted sequence Peak

MA0915.1 dve 12.1441 0.985360610986

NT_033779.5:3824600-

3825000

313 320 - CTAATCCC 2

MA0915.1 dve 11.6975 0.975487844496

NT_033779.5:3824600-

3825000

270 277 + ATAATCCC 2

MA0915.1 dve 11.3993 0.968895149107

NT_033779.5:3824600-

3825000

183 190 - GTAATCCG 2

Table 5 JASPAR - slp 2 - slp1- Analysis Results

Matrix ID Name Score Relative score Sequence ID Start End Strand Predicted sequence Peak

MA0458.1 slp1 10.4209 0.904717113268

NT_033779.5:3833900-

3834100

84 94 - CTGTTTACATG 1

MA0458.1 slp1 11.992 0.943959392058

NT_033779.5:3835455-

3835655

181 191 - TTGTTTTCACA 2

4 CONCLUSION

In this study, kuk, slp1 and slp2 were used as

representatives of class 1 and class 2 genes to

investigate the role of Zld in regulating the expression

of different types of genes. Specifically, Zld plays a

decisive role in the expression of class 1 gene, as

when Zld being knocked out, the expression of kuk

will be greatly reduced; The effect of Zld on class 2

gene is not direct and definite because the expression

of slp1 and slp2 don’t witness such significant

decrease after same procedure. These findings further

confirm that zin-finger protein Zld plays an important

role in drosophila embryonic development.

At the same time, this study comes across two

tentative conclusions for further researches to

confirm. First, several transcription factors (i.e. Dve,

Kr, Hb, etc.) were identified that might collaborate

with Zld to control the expression of pair-rule gene.

Secondly, slp 1 is a potential enhancer of slp2. The

data in this paper support these hypothesis, but

uniquely designed experiments are needed to

validate.

ACKNOWLEDGMENT

We thank Doctor Christine Rushlow for the guidance

and her lab for providing the research data used in this

paper.

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