Regulation of ecmF gene expression and genetic hierarchy among STATa, CudA, and MybC on several prestalk A‐specific gene expressions in Dictyostelium

Abstract

STATa, a Dictyostelium homologue of metazoan signal transducer and activator of transcription, is important for the organizer function in the tip region of the migrating Dictyostelium slug. We previously showed that ecmF gene expression depends on STATa in prestalk A (pstA) cells, where STATa is activated. Deletion and sitedirected mutagenesis analysis of the ecmF/lacZ fusion gene in wild-type and STATa null strains identified an imperfect inverted repeat sequence, ACAAATANTATTTGT, as a STATa-responsive element. An upstream sequence element was required for efficient expression in the rear region of pstA zone; an element downstream of the inverted repeat was necessary for sufficient prestalk expression during culmination. Band shift analyses using purified STATa protein detected no sequence-specific binding to those ecmF elements. The only verified upregulated target gene of STATa is cudA gene; CudA directly activates expL7 gene expression in prestalk cells. However, ecmF gene expression was almost unaffected in a cudA null mutant. Several previously reported putative STATa target genes were also expressed in cudA null mutant but were downregulated in STATa null mutant. Moreover, mybC, which encodes another transcription factor, belonged to this category, and ecmF expression was downregulated in a mybC null mutant. These findings demonstrate the existence of a genetic hierarchy for pstA-specific genes, which can be classified into two distinct STATa downstream pathways, CudA dependent and independent. The ecmF expression is indirectly upregulated by STATa in a CudAindependent activation manner but dependent on MybC, whose expression is positively regulated by STATa.

Analysis of DrkA kinase’s role in STATa activation

“…These are as follows: (a) The β‐galactosidase staining showed that drkA gene is selectively expressed in prestalk A (pstA) cells, where STATa is activated (Figure b). In particular, during the slug–early culminant stages, strong expression was detected in tip‐organizer cells, where the cudA gene, a known STATa target gene (Fukuzawa & Williams, ), and several other potential STATa target genes, such as ecmF and mybC (Saga et al, ; Shimada et al, ; Shimada et al, ), are expressed. (b) Tet‐off induced transient over‐expression of DrkA‐Myc upregulated the phosphorylation level of STATa on Tyr702 compared to the DrkA‐Myc uninduced cells, independent of cAMP addition (Figure b).…”

“…RNA isolation and semi‐quantitative reverse transcription‐polymerase chain reaction (PCR) were largely performed as described (Saga et al, ; Shimada, Inouye, Sawai, & Kawata, ). Total RNA was purified with the RNeasy Plus Mini Kit (QIAGEN) and treated with DNase I (RNase‐Free DNase Set; QIAGEN) followed by purification with the RNeasy MinElute Cleanup Kit (QIAGEN).…”

“…These morphogenetic defects could be attributed to the loss or impaired function of the developmental organizer, which is located in the tip region of the migrating slug. Indeed, the STATa null strain underexpresses various organizer marker genes, including ones encoding several important transcription factors, such as cudA and mybC (Fukuzawa & Williams, ; Saga, Inamura, Shimada, & Kawata, ; Shimada, Maeda, Urushihara, & Kawata, ; Shimada, Nishio, Maeda, Urushihara, & Kawata, ). STATa is activated (i.e., phosphorylated at Tyr702) when extracellular cAMP, which binds the serpentine‐type receptor cAR1, is added to cells in buffered culture conditions (Araki et al, ).…”

The cytosolic lncRNAdutA affects STATa signaling and developmental commitment in Dictyostelium

“…One strain harbored the approximately 300-bp act15 promoter fused to the short dutA promoter to drive dutA expression (we designated this transformed strain OE, meaning a simple overexpressor). The other strain harbored the organizer-specific ecmF promoter (Saga et al, 2016) fused to the short dutA promoter to drive dutA expression (we designated this transformed strain EOE, meaning an ectopic overexpressor).…”

Two potential evolutionary origins of the fruiting bodies of the dictyostelid slime moulds

“…The high cAR activity in the sorogen tip also leads to nuclear translocation of the transcriptional regulator signal transducer and activator of transcription a [STATa, also called Dictyostelium STAT A (DstA)] in prestalk cells (Araki et al , ; Verkerke‐van Wijk et al , ). STATa controls the expression of several genes, including the transcription factors ‘culmination defective’ A (CudA) and MYB domain‐containing C (MybC) (Fukuzawa & Williams, ; Saga et al , ). Intracellular cAMP, on the other hand, activates protein kinase A (PKA).…”